LABORATORY MANUAL CUM PRACTICAL RECORD For B. Tech (Dairy Technology) Degree

GENERAL MICROBIOLOGY (Course No: DM 111)

By

Dr. Velugoti Padmanabha Reddy, Ph.D Professor and University Head

& Dr. I. Sankara Reddy, Ph.D

Professor Dept of Dairy Microbiology

College of Dairy Technology Tirupati-517 502

COLLEGE OF DAIRY TECHNOLGY

SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI- 517 502

2006

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DAIRY MICROBIOLOGY LABORATORY

COLLEGE OF DAIRY TECHNOLOGY TIRUPATI-517 502 A.P

CERTIFICATE

Certified that this is a bonafied record of practical work done by Mr./Mrs.___________________________ ___________I.D. No__________ of I Year B. Tech (Dairy Technology) in the Course No DM 111, General Microbiology during the Year ________________

Signature of the Course in charge

Signature of the Examiner

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GENERAL INSTRUCTIONS FOR WORKING IN A MICROBIOLOGICAL LABORATORY

While working in a microbiological laboratory it is very much essential to maintain absolute cleanliness to prevent microbial contamination from various sources such as equipment, air and working person etc. and hence it is important that the following instructions are always followed very scrupulously.

1. Always wear white, clean laboratory coat or an apron

2. Keep the work area neat and clean at all times

3. Everyday before commencing and after completing the laboratory work clean the top of the work bench with a detergent and followed by a disinfectant ( 5% phenol, Hypochlorite olution containing 200 to 300 ppm available chlorine, 0.5 to 1% formaline etc.,)

4. Before starting and after completing the work wash the hands soap and water. If the handling of the cultures is required wash hands with mild disinfectant (eg. Hypochlorite solution containing 50 to 100 ppm available chlorine)

5. Follow personal hygiene in the microbiological laboratory and do not cultivate the habit of biting the fingers, pens etc., and moistening the labels with tongue as there is a risk of infection with pathogenic microorganisms.

6. Handle all microbial cultures carefully. If a tube, petri dish with culture is broken, pour disinfectant solution and remove the contents carefully. Clean the area again with suitable detergent and disinfectant

7. Handle all the glassware and equipment carefully avoiding damage or breakage. Sterilize the inoculation loop before and after its use.

8. All the used glassware like petri dishes, test tubes, pipettes etc are placed in a separate container provided for that purpose. At no circumstances the used material is left scattered on the workbenches.

9. Whenever the microscope is used, it should be properly cleaned both before and after use. Always use only tissue papers for cleaning lenses.

10. Read carefully the procedures and keep all necessary reagents, materials at the disposal

before commencing the experiment. 11. Wash the hands and other skin surfaces immediately and thorughly if contaminated with blood

or body fluids 12. When leaving the laboratory see that the reagents, stains and other material used should be

returned to their original position and the electrical appliances, water outlets and gas connection are put off.

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INDEX Sl No

Date

Name of the experiment

Page No

Remarks

1 Familiarization, Use and Care of A Bright Field Microscope

2 Use Of Common Microbiological Equipment

3 Physical And Chemical Methods Of Sterilization

4 Simple staining techniques using direct stain and negative stain

5 Differential staining techniques

6 Examination of motility of living bacteria

7 Measuring the size of different bacterial cells

8 Morphological examination of bacteria

9 Preparation of common microbiological media

10 Techniques of isolation and purification of microorganisms

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Growth and cultural characteristics of microbial cultures

12 Study of biochemical characteristics of bacteria

13 Effect of physical and chemical agents on growth of bacteria

14 Estimation of bacteria and fungi in air

15 Estimation of number and type of organisms in soil

16 Enumeration of microorganisms in water

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I. GENERAL TOOLS AND EQUIPEMENT USED IN A MICROBIOLOGICAL LABORATORY

MICROSCOPY Microorganisms can not be viewed with naked eye and hence special instruments like microscope are essential for studying their morphological characteristics. Generally two types of microscopes are used in most of the laboratories and they are simple and compound microscope. A microscope consists of lens or combination of lenses for enlarging the image of the objects that are difficult for viewing with naked eye. A simple microscope is essentially an ordinary magnifying lens that can magnify an object up to 20 times the size of the object under view. A compound microscope consists of two separate lens systems ( eyepiece and objective) fitted in a body tube of microscope, arrangements for the illumination of the objects and mechanical adjustable parts for determining focal length between lenses and object or specimen. A compound microscope can be a bright field light microscope, Dark field light microscope, Phase contrast microscope, Ultra violet microscope etc., A bright field light microscope is used for routine microbiological work and others are used for special purposes. Exercise 1: Familiarization, Use and Care of A Bright Field Microscope Aim: To familiarize the students with components of compound microscope and with and care of compound microscope Major parts of a compound microscope: 1. Stage: It is a fixed platform with an opening in the center for allowing the

passage of light from an illuminating source. This platform provides a surface for the placement of slide containing the object and facility for proper positioning of the slide with the help of clips and for moving the slide horizontally in two directions

2. Illumination: The ordinary day light or artificial light can be used as a source

of illumination. The light from illumination source is reflected into condenser via mirror. This is a mirror with one side plane and other side concave. The plane or flat side of the mirror is used for artificial light and the concave side is used for sunlight. Some microscopes are fitted with built in lamp and in such cases the mirror is not required as the light directly passes through condenser

3. Condenser: It is directly found under the stage and contains two sets of

lenses that collect and concentrate light that is reflected into the condenser

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from light source. It is provided with an iris diaphragm which regulates the amount of light entering the condenser.

4. Body tube: It is found above the stage of the microscope. It consists of lens

system that magnifies the object. Through this the light passes from the objective to the eyepiece. It consists of eyepiece lens and objective lens. Eyepiece or ocular lens is housed in the upper part of the tube while the lower portion consists of a movable nosepiece containing objective lenses. The nose piece can be revolved so as to bring any required objective into proper alignment for viewing the specimen.

5. Lenses: The magnification of the microscope depends on the ocular and

objective lens systems. The total magnification of a microscope is equal to the product of ocular lens and objective lens magnification For eg. If magnification of objective lens is 40X and of ocular is 10X then the total magnification is equal to 400X Objective lenses: Generally the compound microscope consists of three objective lenses i.e low power (focal length 16 mm), High power(focal length 4 mm) and oil immersion objective (focal length 1.8 mm). As the magnification increases the amount of light required is also increased. Eyepiece or Ocular lens: Viewing of the object is made through this lens. Depending upon the magnification of ocular lens it magnifies the image formed by the objective lens by 5 to 15 times.

6. Knobs: These knobs can be operated basing on the screw mechanism

Coarse adjustment knob: The knob is used to bring the object approximately into focus. Fine adjustment knob: The knob has a limited range and is used to bring the object exactly into focus after the coarse adjustment has been made. Sub-stage adjustment knob: It is used to raise or lower the condenser for regulating the light coming to the object.

Use and care of the microscope 1. Place the microscope firmly on a laboratory bench and clean the objective and ocular lenses thoroughly with the help of acceptable lens tissue. 2. Turn the plain side of the mirror towards the sunlight and incase of a lamp as

the source of light turn the concave side of the mirror towards lamp to reflect maximum light.

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3. Place the microslide with specimen within the clips on the stage of the microscope. Move the slide on the stage in such a way that the smear comes directly over the condenser.

4. Rotate the low power objective into position. Lower the body tube with coarse

adjustment knob to its lowest position i.e. about a quarter inch above the glass slide.

5. Look through eyepiece and slowly raise the stage with coarse adjustment knob until the smear comes into focus. 6. Adjust the sub-stage condenser and iris diaphragm to produce optimum

illumination. Sharpen the focus with the help of fine adjustment knob. 7. Swing the high-power into position and with the help of fine adjustment knob

sharpen the focus. Most of the microscopes are constructed in such a fashion that once the low power is in focus the next higher powers will have same focal length and can be brought into focus only with fine adjustment knob.

8. Now remove the slide from the stage and place a drop of immersion oil on the smear to be viewed and rotate the nosepiece until the oil immersion locks

into position Now look through the ocular lens and fine adjustment knob is readjusted to

bring the image into sharp focus. Always focus while raising the tube and never while the tube is lowered.

9. Observe the size, shape and arrangement of cells appearing in a number of

microscopic fields. 10. After completing the observations remove slide and clean the objective

lenses with lens tissue moistened with xylene and again wipe with dry lens tissue

Questions: 1. Draw a neat sketch of a microscope and label all parts? 2. What are the ways of enhancing the resolving power? 3. Define the term parafocal of a microscope? 4. What is the purpose of using cedar wood oil with oil immersion lens?

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Exercise 2 : Use Of Common Microbiological Equipment In a microbiological laboratory, a number of special types of instruments and equipment described below are necessary for the analysis of samples and maintenance and study of microbial cultures Aim: To familiarize the students with common microbiological equipment and with their use and care 1. Hot air oven: This is useful for the sterilization of glassware, fats, oils and

powders. It is a rectangular chamber having double walls insulated with asbestos or glass wool on all sides. The equipment is provided with heating coils in side walls and at the bottom for heating. A thermometer with a recording capacity of more than 2000C is placed on the top and the unit is provided with a thermo-regulator to control the temperature. A temperature of 1600C to 1800C is used for atleast one to one and half hour

2. Autoclave: The autoclave is useful for the sterilization of liquid, semi-solid

media, Glassware containing discarded cultures, rubber materials, aprons, syringes etc.,. It operates on the principle of pressure cooker using steam under pressure. Usually the operating conditions at 15-lbs. pressure for 15 minutes are sufficient to give complete sterilization of material by killing all bacteria and their spores, as these conditions will accomplish a temperature of 1210C. It is more effective than dry heat used in hot air oven because the steam has better penetration power and denatures protein at a lower temperature than dry heat. The temperature can be increased by increasing the pressure and decreased by decreasing the pressure. The instrument is provided with a pressure gauge and safety valve. The valve when it is open, allows steam to go out and when closed controls it by allowing steam to escape slowly so that a constant pressure is maintained.

3. Serum inspissator: This is useful for the sterilization of materials like media containing blood serum by employing moist heat at low temperature. The method employs a temperature of 800C for two hours on three successive days. It consists of double jacketed water bath suitably insulated and provided with specially designed racks to keep the media tubes in slanting position. It is heated with the help of immersion heater and the temperature is controlled by a thermostat.

4. Arnold steam sterilizer: It is meant for sterilization of materials like media

containing sugars, milk, gelatin etc., which require less than 1000C. The sterilization is accomplished with the help of flowing steam at a temperature of 1000C for 30 minutes. However, it will not be sufficient to kill spores and hence fractional sterilization or tyndallization for three successive days is recommended for complete sterilization.

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5. Incubator: It is useful in maintaining constant optimum temperatures necessary for the growth of microbial cultures. It is an insulated chamber having different compartments and is provided with heating devices such as coils at the bottom and a thermostat for maintaining the temperature.

6. Laminar flow chamber: This unit is used for controlling microflora in closed

spaces like cabinets. The unit acts as sterile workbench in which air passes through high efficiency particulate air filters (HEPA) consisting of cellulose acetate as filter medium pleated around aluminum foil. It is efficient in removing particles as small as 0.3 m as air passes through a bank of these filters and into the enclosure so that entire body of air moves with uniform velocity along parallel flow lines. There are a variety of designs of these units such as vertical or horizontal flow systems. The unit also comprises of a UV lamp for additional sterility of workplace.

7. Filters: The seitz filters and membrane filters are very useful for sterilization

of heat sensitive biological fluids but the fluids must be relatively free of suspended particulate matter. The membrane filter unit consists of a receiving flask, filter base and filter support. In membrane filters the pores are of uniform and specific, predetermined size and they are composed of biologically inert cellulose esters. They are prepared as circular membranes of about 150 m thickness and contains millions of microscopic pores of a very uniform diameter. The porosity can vary from approximately 0.01 to 10 m but for the microbiological purposes the filters with 0.45 m pores are used.

Questions: 1. What is the use of a BOD incubator? 2. What is the principle of Tyndalization? 3. Mention various other equipment commonly used in microbiological laboratory?

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II. METHODS OF STERILIZATION

The microorganisms are ubiquitous and are found in almost all situations exposed to the atmosphere. During microbiological work involving the isolation, propagation and maintenance of pure cultures it is very much essential to eliminate the existing and extraneous microbes from media, glassware and other material that are used in the laboratory. Sterilization is the process of destruction of all types of microbial life including bacteria, spores and viruses. The destruction of these organisms can be accomplished by means of physical and chemical methods. The physical methods involve the application of dry heat, moist heat and filtration, where as in chemical methods a number of chemical substances such as alcohols, aldehydes, phenols, chlorine compounds, gaseous substances ( ethylene oxide, formaldehyde vapours etc.,) are used Exercise 3: Physical And Chemical Methods Of Sterilization Aim: To study the physical and chemical methods of sterilization of various laboratory materials including media

I) Physical methods: A) Sterilization by using dry heat: This is useful for the sterilization of glassware, fats, oils and powders by using dry heat. The temperatures generally employed are 160oC for 1 hour, but incase of heavy loads or for oils and powders a temperature of 160oC for 2 hours is followed. Materials: 1. Hot air oven 2. Materials for sterilization like glassware Procedure: 1. The glassware meant for sterilization must be clean and dry 2. Wrap the glassware in paper or place them in suitable metal container made

up of copper or stainless steel 3. Place the material in the hot air oven without over packing the space. 4. Close the door tightly before setting the temperature knob in on position.

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5. Maintain temperature at 160oC and keep the material at this temperature for one hour

6. Disconnect the electrical connection or switch off the temperature knob 7. Allow the temperature to come down before taking material for use,

otherwise the glassware may crack B) Sterilization by moist heat: In this method the sterilization is attained by using moist heat at temperature above 100oC by the application of saturated steam under pressure. The vessel used for this purpose is known as autoclave. A pressure of 15 psi achieves a temperature of 121°C which is sufficient to sterilize the material in 15 min. Materials: 1. Autoclave 2. Materials for autoclaving like media, gloves etc., Procedure: 1. Place the material for sterilization in the autoclave and close the lid or door of

autoclave and switch on electrical connection. In case of non-jacketed autoclave, see that there is sufficient amount of water in the chamber for generation of steam.

2. Open the steam escape valve till steam starts escaping from the steam

chamber. It is very important to displace all the air from autoclave since the pressure of the air results in lower temperature at a given pressure and air also may settle at the bottom of the autoclave forming an unheated layer there.

3. Close the steam valve and allow the pressure to raise slowly till the desired temperature and pressure are reached.

4. Maintain the desired temperature and pressure for desired period of time.

This may vary between 10 to 20 minutes depending upon the material for sterilization and pressure or temperature of the autoclave.

5. Put off the electrical connections and open the steam valve slowly to permit

the autoclave to cool down until the pressure gauge reads zero 6. Open the door of the autoclave and remove the sterilized material for use.

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C) Sterilization by filtration: Filtration is used as sterilization method for some materials like carbohydrate solutions, serum etc. which can not be sterilized by dry heat or moist heat. Materials: 1. Seitz filter -containing asbestos pads. 2. Material for sterilization such as glucose solution. Procedure: 1. Assemble the apparatus by fitting asbestos disk into a special metal container

called seitz filter. The asbestos filter pads are disk shaped and vary in size and grades of porosity.

2. Below the asbestos disk place metal screen that comes along with the

apparatus. 3. Place the screen and the disk in metal container and then tighten by means of

set screws 4. Cover the entire apparatus in aluminum foil and then tightly fit into the

opening of filter flask. Plug the arm of the filtering flask with non-absorbent cotton.

5. Sterilize the whole apparatus in an autoclave at 15 psi for 30 minutes. 6. Attach the apparatus to the vacuum pump 7. When the filtration begins, if foam is forming around asbestos disk, simply

tighten the screws that hold it in metal shield. 8. After use the asbestos disks are disposed off in suitable manner either by

burning or autoclaving

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II. Chemical methods Certain chemicals, which are toxic to microorganisms, can be used as sterilizing agents. The most common chemical sterilization methods used are fumigation and the use of alcohol in the laboratory. Fumigation: Materials: 1. Potassium permanganate 2. Formalin 3. Suitable container Procedure: 1. Calculate the space of the room or the incubator to be sterilized in terms of

cubic feet. 2. Add sufficient quantity of Potassium permanganate to the container and pour

sufficient quantity of formalin on it. Usually 250 grams of Potassium permanganate and 500 ml of formailn are sufficient to fumigate 1000 cubic ft. space.

3. Immediately place the container in the room or in the incubator to be

fumigated and close the door air tight. 4. After 24 hours of fumigation open the door of the room or the incubator and

remove the container used for placing the two reacting chemicals. 5. The space can be used, once it is cleared of the fumes. Questions: 1. How is the moist heat more effective than dry heat? 2. What is the operative principle of fractional sterilization? 3. What is the relationship between pressure and temperature in an autoclave? 4. What are the materials that have to be filter sterilized? 5. Mention various chemicals that can be used for sterilization?

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III. MICROBIAL STAINING METHODS

Visualization of microorganisms in the living state is most difficult because they are very minute, transparent and practically colorless when suspended in an aqueous medium. With the help of staining methods it will be easier to study the properties of microorganisms such as shape, size and structure and to differentiate them into specific groups for diagnostic purposes. A stain (dye) is an organic compound containing 'cromophore' ( a chemical group that imparts colour to the benzene) and 'auxochrome' (chemical compound which imparts the property of ionization of chromogen and binds to fibers and tissues) linked to a benzene group. The action of the dye is both physical and chemical in nature. The ability of a stain to bind to macromolecular cellular components such as proteins and nucleic acids depend on the electrical charge found on the chromogen as well as cellular components to be stained. The stains are either acidic or basic. The acidic dyes are anionic and on ionization the chromogen portion of the stain exhibits a negative charge and has a strong affinity for positive constituents of cell like proteins. The basic stains are cationic in nature and on ionization the chromogen portion of the stain exhibits a positive charge and has a strong affinity for negative constituents of cell like nucleic acids Numerous staining techniques are available for visualization, differentiation, and separation of bacteria in terms of morphological characteristics and cellular structures. These methods fall into two groups viz., simple staining methods and differential staining methods. Exercise 4: Simple staining techniques using direct stain and negative stain Staining procedure that use only one stain is called as simple staining method. A simple stain that stains the bacteria is called a direct stain and a simple stain that stains the background leaving bacteria unstained is called a negative stain Aim : To study the performance of simple staining and negative staining procedures for studying the morphological shapes and arrangements of bacterial cells A. Simple staining with direct stains Materials: 1. Clean, grease free slides 2. Platinum loop and inoculating needle 3. Methylene blue stain (see annexure) 4. Microscope 5. Bunsen burner

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6. Broth and agar cultures of bacteria e.g. Staphylococcus aureus, Lactococcus lactis ssp.lactis, Lactobacillus acidophilus

Procedure: i) Preparation of smear : 1. Take a clean, grease free glass slide and pass it through the Bunsen flame

two or three times to dry it and remove traces of any grease 2. Sterilize the platinum loop in the flame to redness and allow it to cool. 3. In case of a broth culture transfer a loopful of a well shaken broth culture to

the slide and spread it evenly to get a thin and uniform smear 4. For the bacterial culture on solid media, place 1 or 2 drops of distilled water in

the center of the slide using inoculation loop. With the help of a sterilized loop scrape a small amount of culture from solid medium without gouging the agar. Emulsify the culture in the drop of water and spread the suspension to make a thin smear.

5. Allow the smears air-dry and then fix the smear by passing the slide rapidly two or three times through the flame with the smear side up. The slide should not be too hot when felt by the back of the palm

ii) Staining the smear: 1. Place the slide, with smear, on the staining tray and flood the smear with

either of the methylene blue or crystal violet stain and leave for 30 to 60 seconds.

2. Carefully wash off the excess stain with distilled water by letting the water

down the tilted slide. 3. Dry the smear by gentle pressing of it with clean blotting paper or allow it air

dry. 4. Examine the stained smear microscopically using the low power, high power

and oil immersion objectives. 5. Record your observations with labeled drawings. B. Negative staining : The practical application of negative staining is of two folds. The natural size and shape of cells can be observed because heat fixation is not followed and the

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cells are not subjected for distorting effects of chemicals. It is possible to observe some bacteria such as spirilli that are difficult to stain. Materials: 1. Clean, grease free slides 2. Platinum loop and inoculating needle 3. Nigrosine (see annexure) 4. Microscope 5. Bunsen burner 6. Broth or Agar cultures of bacteria e.g. Bacillus cereus, Micrococcus luteus Procedure: 1. Place a small drop of nigrosine at the one end of the clean, grease free slide. 2. For agar cultures add a loopful of distilled water and emulsify a small amount

of the culture in the nigrosine-water drop and for broth cultures mix a loopful of the broth culture into the nigrosine drop.

3. Using the edge of another glass slide held at a 30 degree angle spread the

drop out to form a thin smear. 4. Air dry the smear. Do not heat fix the smear 5. Examine the stained smear microscopically using the low power, high power

and oil immersion objectives. 6. Record your observations with labeled drawings Questions: 1. What is the purpose of fixing the cells before staining? 2. What could be the reason for the size of the bacteria being more accurate in

negative stain than in simple stain? 3. In addition to nigrosine what are the others stains that can be used for

negative staining?

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Exercise 5: Differential staining techniques The differential staining methods require at least three chemical reagents for staining a heat fixed bacterial smear. The three chemicals, which are used in a sequential order are: a) Primary stain- to impart its colour to all cells, b) Decolorizing agent- to establish a color contrast by retaining or removing the primary stain depending on the chemical composition of cellular components and c) Counter stain- which has contrasting colour to that of primary stain. If the primary stain is not washed off or the cell has retained primary colour even after decolurization, the counter stain will not absorbed, otherwise the counter stain gives contrasting colour to the cellular components. The different methods of differential staining fall into categories like Gram's staining, spore staining, capsular staining, acid fast staining etc., A) Gram's Staining: Gram stain is a differential stain and it allows to group the bacteria into Gram positive and Gram negative. The bacteria are stained purple with primary stain of crystal violet and mordant Gram's iodine intensifies the ionic bond between primary stain and the bacteria. When decolourizing agent ethyl alcohol is used some of the bacteria looses the primary stain while others retain. When secondary or counter stain, safranin, is used the decolorized cells again get stained to look purple. The organisms that retain primary colour are termed as Gram positive and those decolourize but stain with counterstain are termed as Gram negative.

Materials 1. Crystal violet (see annexure) 2. Gram's iodine (see annexure) 3. Ethyl alcohol (95%) 4. Safranin (see annexure) 5. Grease free microslides

Procedure a) Preparation of smear: Prepare the bacterial smear as described earlier in Exercise 4(A) b) Staining of smear: 1. Place the slide with smear on the staining tray and cover the smear with

crystal violet and leave it for 60 seconds

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2. Wash the slide carefully with distilled water from a wash bottle 3. Without drying the smear cover it with Gram's iodine and leave it for 60

seconds. Wash the slide gently with water 4. Decolorize with alcohol by adding drop by drop at one corner of the slide and

allowing it to run over the stained smear for 30 seconds. 5. Cover the smear with safranin and leave it for 30 seconds. Wash the slide

gently with water. 6. Blot dry the smear with blotting paper or air dry 7. Examine the smear under microscope. B) Spore staining: The important groups of bacteria forming spores are Bacillus sp. And Clostridium sp. These organisms under certain unfavourable conditions develop into spores, which are refractile bodies and resistant to physical and chemical agents. The spores are difficult to stain and but once stained they are difficult to decolurize. Malachite green and safranin are generally used for spore staining.

Materials:

1. 5.0% aqueous solution of malachite green 2. 0.5% aqueous solution of safranin 3. Grease free microslides 4. Inoculating loop 5. Cultures of Bacillus subtilis

Procedure: 1. Prepare a smear of the culture and fix it as described earlier in Exercise 4 (A) 2. Cover the smear with malachite green solution and allow for 30 to 60

seconds. Heat the slide by holding it over a low flame until the stain begins to boil. Allow the heating for 5 minutes taking care that the stain does not dry up. Replenish the stain as needed..

3. Wash the slide in water and air dry (water acts as decolourizing agent) 4. Counter stain with safranin solution for 30 to 60 seconds. 5. Wash the slide in water and air dry

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6. Examine the smear under oil immersion objective of the microscope C) Capsular staining: A capsule is a gelatinous outer layer secreted by the cell that surrounds and adheres to the cell wall. Capsular material is water-soluble and may be dislodged and removed from vigorous washing. Smears also should not be heated as the resultant cell shrinkage may create a clear zone around the organism and this artifact can be mistaken for the capsule. Because of the nonionic nature the simple stains will not adhere to the capsule. Most of the capsular stains will stain the bacteria and the background leaving the capsules unstained i.e. essentially a negative capsular stain.

Materials: 1. Crystal violet (1% aqueous solution) 2. Copper sulfate (20% aqueous solution) 3. Grease free microslides 4. Inoculating loop 5. Cultures of Alkaligenes viscosus, Enterobacter aerogenes Procedure: 1. Prepare a thick smear of the culture and let it air dry. Do not heat fix the

smear. 2. Flood the smear with crystal violet and leave it for 5 to 7 minutes. 3. Wash the smear several times with copper sulfate solution 4. Blot dry the smear with blotting paper or air dry 5. Examine the smear under oil immersion objective of the microscope D) Acid fast staining: The cell walls of certain bacteria like species belonging to genus mycobacterium contain a wax like lipid called mycolic acid, which renders the cell wall impermeable to most of the stains. Once the stain has penetrated it can not be readily removed even with vigorous use of acid alcohol as decolurizing agents. This property makes these organisms to be termed as Acid- fast while others as non-acid-fast.

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Materials: 1. Concentrated carbol fuschin (see annexure) 2. Acid-alcohol ( mix 97ml of 95% Ethyl alcohol with 3 ml of con.HCl ) 3. Alkaline methylene blue ( 0.3% aqueous solution) 4. Microslides 5. Culture of Mycobacterium tuberculosis Procedure: 1. Prepare the smear and heat fix as described earlier in Exercise 4(A) 2. Apply carbol fuschin to the smear and heat for 5 minutes. Donot allow the

stain to evaporate 3. Wash the slide with distilled water 4. Add acid-alcohol drop by drop until the alcohol runs clear for 15 to 20

seconds. 5. Wash off the acid-alcohol with distilled water 6. Counter stain with methylene blue for 30 to 60 seconds 7. Wash the slide in water and air dry 8. Examine the smear under oil immersion objective of the microscope Questions: 1. What is the advantage of Gram's stain over the simple stain? 2. Why is the age of the culture is important in a Gram's stain? 3. Name two Gram positive and Gram negative organisms? 4. What is the difference between a vegetative cell and a spore? 5. Give the types of culture media that would increase the size of a bacterial

capsule? 6. What are the diseases that can be diagnosed by employing acid-fast

procedure?

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IV. MOTILITY OF BACTERIA Bacteria are unicellular and are widely distributed in the nature. Some species of rod shaped bacteria such as Bacillus subtilis, Pseudomonas fluorescence, Escherichia coli are motile by means of flagella. This test of motility will be useful during characterization of the unknown species of bacteria. Exercise 6: Examination of motility of living bacteria Aim : To examine the living bacteria for their motile character using hanging drop

method and motility medium method A) Hanging drop method: Materials: 1. Microslides with depression 2. Cover slips 3. Platinum loop 4. Microscope 5. Vaseline 6. Broth cultures of organisms Bacillus subtilis, Pseudomonas fluorescence,

Escherichia coli Procedure: 1. Take a clean microslide with depression and apply Vaseline around the

depression. 2. Place a loopful of broth culture ( preferably overnight grown) on the center of

the cover slip 3. Invert the microslide and place it on the cover slip in such a fashion that the

cover slip adheres to the slide forming a chamber, with the droplet of culture in it.

4. Quickly turn the slide upside down so that the cover slip is on the top with the

droplet of the culture hanging down in depression. 5. Transfer the slide with cover slip to the stage of microscope and examine

under low power. Now taking care not to break cover slip, change to high power and also to oil immersion objectives for examining the cells in droplet. Adjust the light, by lowering the condenser or partly closing the iris diaphragm and/or both

6. Observe for the difference in the movements of cells of different organisms.

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B) Motility Agar medium method Materials: 1. Motility test medium Tryptose 10.0 g Sodium chloride 5.0 g Agar 5.0 g Distilled water 1000 ml 2. Broth cultures of organisms Bacillus subtilis, Pseudomonas fluorescence,

Escherichia coli Procedure: 1. Rehydrate the medium and heat gently into solution. Transfer approximately 5

ml of medium into test tubes and sterilize by autoclaving at 1210C for 15 minutes.

Allow the medium to cool in an upright position and refrigerate for storage 2. Stab the center of the medium in tube with pure culture (grown for 18-24 hr)

using a inoculating needle to a depth of 1/2 inch 3. Incubate the tubes at 350C for 24 to 48 hrs. 4. Observe for the motility of the culture. If it is positive for motility the organism

migrates from the stab line and diffuses into medium causing turbidity and may exhibit fuzzy streaks of growth. If it is non-motile the growth accentuated along the stab line with surrounding medium remaining clear.

5. If negative incubate the tubes at 21-250C for 5 days and observe for motility

as described above. Questions: 1. What is a wet mount method and how does this differ with hanging drop

method? 2. Differentiate bacterial motility and Brownian movement? 3. Why is the age of the culture is important in testing the motility of an

organism?

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V. MEASUREMENT OF THE SIZE OF MICROORGANISMS The bacterial cells are usually measured in microns and viruses in millimicrons. The rod shaped bacteria are measured in length and thickness while cocci are usually measured in diameter. The cocci vary in size from 0.5 to 1 m in diameter and rod shaped bacteria from 0.2 to 0.6 m in thickness and 0.5 to 6.0 m in length. Exercise 7: Measuring the size of different bacterial cells Aim: To perform the microscopic examination for measuring the size of bacteria. Materials: 1. Microscope 2. Ocular micrometer 3. Stage micrometer 4. Stained smears of bacterial cultures Procedure: 1. The ocular micrometer is carefully placed into the eyepiece of the

microscope. The graduations on this ocular micrometer are arbitrary and must be calibrated for

each eyepiece. 2. Place the stage micrometer on the stage of the microscope. The length of the

scale on this stage micrometer is 1 millimeter .Since there are 100 divisions on the scale, each division equals to 0.01 mm or 10 m

3. First focus the stage micrometer under the low power objective and then

slowly rotate the eyepiece to superimpose the graduations of ocular micrometer over those of stage micrometer.

4. Without disturbing microscope, place a drop of immersion oil on stage

micrometer and focus under oil immersion objective by making fine adjustment, if necessary.

5. Move the mechanical stage of the microscope slowly so that a line of the

stage micrometer coincides with a line on ocular micrometer. Next find another line of stage micrometer that coincides with next line of ocular micrometer and note the number of divisions on stage micrometer and corresponding number of coinciding divisions on ocular micrometer.

6. Calculate calibration factor for one ocular division as follows:

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Calibration factor for Known distance between 2 lines of stage micrometer Ocular division (mm) = Number of divisions coinciding on ocular micrometer 7. Remove the stage micrometer and place the stained smear of bacterial

culture under oil immersion objective. 8. Calculate the number of ocular divisions that a bacterial cell occupies 9. Determine the size of the bacterial cell as : Size in m = No. of ocular divisions occupied by bacterial cell X Calibration

factor for one division of ocular micrometer Questions: 1. What is the need of calibration of the ocular micrometer? 2. Mention the sizes of various important bacteria?

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VI. MICROSCOPIC EXAMINATION OF MICROORGANISMS

Microorganisms are morphologically different. The morphological examination will be helpful in identification of the microorganisms. This includes studying the stained smears for shape, size, arrangement of cells and for cellular structures and also for the motility using hanging drop method. Exercise 8: Morphological examination of bacteria Aim: To perform microscopic examination for studying the morphological

characteristics of bacteria Materials: 1. Microscope 2. Microslides 3. Staining solutions 4. Nutrient agar slants of different bacterial cultures Procedure: 1. Test the cultures using hanging drop method for motility 2. Prepare the smears of different organisms. Fix the smears and stain using

different staining techniques such as Gram's staining, Capsular staining, spore staining etc.,

3. Examine the smears for morphological characteristics under oil immersion. 4. Observe, make a sketch and describe the organism using the following

terms:

A) Shape: Spherical, short or long rods, coccobacillary, spiral, filamentous B) Size: Size is expressed in m. Spherical shaped organisms are

measured in diameter and rod shaped organisms in length and breadth C) Arrangement: Single, in twos, in groups of four or eight, in grape like

bunches, in short or long chains, scattered, bundles or in any other form D) Sides: Straight, concave, bulging, parallel or irregular E) Ends: Round, Pointed, truncate or concave F) Capsule: Present or absent and if present the size of the capsule

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G) Flagella: Atrichous, monotrichous, amphitrichous, or peritrichous

H) Spores: Spherical or oval. Location is central, subterminal or terminal in

the cell

I) Reaction to Gram's stain: Gram positive or negative

J) Pleomorphism: The deviation annd variation in size and shape of the organism may be observed

K) Motility: Present or absent

Questions: 1. Apart from morphological characteristics what are the other parameters

required to identify the organisms 2. How do the yeast and bacteria differ in shape and size?

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VII. PREPARATION OF MEDIA The survival and continued growth of the microorganisms depends on an adequate supply of nutrients apart from a favourable growth environment. It is therefore essential to provide various soluble low molecular weight substances that are generally derived from enzymatic degradation of complex nutrients for the growth of microbes. The medium that contains all these ingredients required for the growth of microorganisms is know as culture medium. A liquid medium lacking a solidifying agent is called as broth and a broth supplemented with a solidifying agent (agar) results in a solid or semi solid medium. The solid medium can be used in the form of agar slants, agar deep tubes and agar plates depending upon the need. The media can be simple media or differential media. A simple medium generally supports the growth of the common microorganisms but without being able to differentiate them. The differential media contain various nutrients that allow the investigator to distinguish one organism from another by the metabolic change they produce in the medium. MacConkey's agar, Blood agar, Brilliant green bile agar are the certain examples of differential media. Exercise 9: Preparation of common microbiological media Aim: To familiarize with the methods of preparation of common microbiological media. A) Preparation of Nutrient medium: The nutrient medium is the common medium employed for the detrmination of viable bacterial numbers and cultivation of aerobic organisms. It is also a basal medium for a variety of physiological tests. The medium can be used as a liquid medium (Nutrient broth) or a solid medium ( Nutrient agar). The composition of the media includes meat extract which contains water soluble substances like carbohydrates, organic nitrogenous compounds, water soluble vitamins and salts ; Peptone a principal source of nitrogen ; and Sodium chloride to increase salt content Materials: Peptone 5.0 g Meat extract 3.0 g Sodium chloride 5.0 g Agar 15.0 g* Distilled water 1000ml (Final pH 7.2 ) * Agar is eliminated if the nutrient broth is to be prepared.

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Procedure: 1. Weigh the ingredients as per the composition and dissolve in 2/3 quantity of

distilled water by steaming for 10 minutes. 2. Cool the medium and adjust the pH for 7.2 using NaOH solution. Steam again

and filter through cotton pad 3. Adjust the volume to 1000 ml with distilled water. 4. Pour the medium in suitable container like flasks or tubes and sterilize the

contents in an autoclave at 15 lbs pressure for 15 minutes. B) Preparation of Differential medium: This can distinguish among the morphologically and biochemically related groups of organisms. In this medium certain chemicals are incorporated which produce a characteristic change in appearance of the bacterial growth and or the medium surrounding the colonies that permit differentiation. Ex. MacConkey's medium. In this medium the inhibitory action of crystal violet on the growth of gram-positive organisms allows the isolation of gram-negative bacteria. Incorporation of carbohydrate lactose, bile salts and pH indicator neutral red permits differention of enteric bacteria on the basis of their ability to ferment lactose and produce change in pH. Materials: Composition of MacConkey's medium Peptone 20 g Lactose 10 g Sodium chloride 5 g Sodium taurocholate 5 g Neutral red 0.03 g Crystal violet 0.001 g Distilled water 1000 ml (Final pH 7.4 ) Procedure: 1. Weigh the ingredients as per the composition and dissolve in 2/3 quantity of

distilled water by steaming for 10 minutes. 2. Cool the medium and adjust the pH for 7.4 using NaOH solution. Steam

again and filter through muslin cloth

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3. Adjust the volume to 1000 ml with distilled water. 4. Pour the medium in suitable container like flasks or tubes and sterilize the

contents in an autoclave at 15 lbs pressure for 15 minutes Questions: 1. What is the function of bile salt in MacConkey’s medium? 2. What is agar? What is the need of agar in solid media ? 3. What are the different nutrients supplied by a) Peptone b) Meat extract c)

Yeast extract d) Sodium Chloride? 4. What is the reason for lowering of the pH of the medium during heat

sterilization?

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VII. ISOLATION TECHNIQUES FOR MICROORGANISMS In nature, microbial populations do not segregate themselves by species but exist with a mixture of many other cell types. These can be separated into pure cultures by various techniques. Once, discrete well developed colonies develop on the surface of the medium each colony may be picked up with a sterile needle and transferred to agar slants. Each of these new slant cultures represent the growth of a single species of organism and is designated as pure culture or stock culture. Subsequently these may be used for the study of their cultural and biochemical properties. The common methods of isolation of microbes are streak plate method, spread plate method and pour plate method Streak plate technique is a rapid qualitative isolation method. It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate. In this method a loop is used to streak the mixed sample many times over the surface of a solid culture medium in a petri plate. By streaking the loop repeatedly over the agar surface the bacteria fall off the loop one by one and each cell develops into a colony In spread plate technique, a small amount of a previously diluted sample is spread over the surface of a solid medium using a bent glass rod shaped like a hockey stick. In the pour plate technique, multiple dilutions of a sample are mixed with melted agar and poured into petri plates. Some of the plates will have separated colonies due to dilutions. Exercise 10: Techniques of isolation and purification of microorganisms Aim: To familiarize with techniques of isolation and purification of organisms A) Streak Plate method: Materials: 1. Nutrient agar plates 2. Broth of mixed bacterial culture 3. Inoculation loop Procedure: 1. Sterilize the inoculation loop by flaming it to redness , allow it to cool and pick

up a small amount of culture by the loop aseptically near the flame of gas burner

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2. Place the picked up culture on the sterile agar surface. Flame and cool the loop and drag the culture several times across the surface at one side of the petri dish.

. 3. Turn the petri dish 90 degrees and touch the loop to a corner of the culture in

the first streak area and drag for several times across the agar i.e parallel streaks across the first streak.

4. Repeat the streaking 2 to 3 times more across the previous streaks The loop

must not touch any of the previously streaked areas. The flaming of the loop at the each streaking is to effect the dilution of the culture so that fewer organisms are streaked in each area resulting in the final desired separation and thus ensuring the dilution effect and facilitating the growth of the discrete isolated colonies.

5. Incubate the plates in an inverted position for 24 to 48 hours at 37oC B) Spread plate method: Materials: 1. Nutrient agar plates 2. Broth of mixed bacterial culture 3. Inoculation loop 4. L-shaped bent glass rod 5. 95% ethyl alcohol Procedure: 1. Place the bent glass rod into the beaker and add a sufficient amount of 95%

ethyl alcohol to cover bent portion 2. Label all the agar plates at the bottom and with a sterile loop place a loopful

of the culture in the center of the agar plate 3. Remove the glass rod from the beaker and pass it through the flame of

Bunsen burner. With the bent portion of the rod pointing downward allow the alcohol to burn off the rod completely and cool the rod for few seconds.

4. With the help of the glass rod lightly touching the agar surface spread the

culture uniformly over the agar surface (for convenience a petri dish turntable can be used). Immerse the glass rod in alcohol and reflame.

5. Incubate the plates in an inverted position for 24 to 48 hours at 37oC C) Pour plate method:

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Materials: 1. Nutrient agar 2. Sterile petri dishes 3. Sterile 1 ml pipettes 4. Sterile dilution blanks (9 ml) 5. Colony counter 6. Broth of mixed bacterial culture Procedure: 1. Label the 9 ml dilution blanks from numbers 1 to 6 or 8 depending on the need. 2. Keep the sterilized nutrient agar in boiling water to liquefy and then in an water

bath maintained at 45oC to maintain its liquid state. 3. Mix the broth containing the bacterial culture thoroughly to disperse the cells. 4. With a sterile pipette transfer 1 ml of the bacterial culture to the number one

dilution blank. Discard the pipette. Now the sample in the first tube will be making 1 in 10 dilution.

5. Mix the number one dilution blank and with the help of a fresh pipette transfer 1

ml of the sample from number 1 blank to number 2 dilution blank.. The sample now becomes diluted by 100 times i.e 1 in 100

6. Similarly with the help of fresh pipette make other serial dilutions to make 1 in

1000; 1 in 10000 and so on. 7. Transfer 1 ml of each of the dilution samples into sterile petri dishes which have

been previously labeled. 8. Using sterile technique, pour 15 to 20 ml of the molten agar at 45oC into the petri

dishes. Mix the contents by rotating each petri dish to ensure uniform distribution of cells in the medium.

9. Allow the agar to solidify and incubate the plates in an inverted position for 24 to 48 hours at 37oC Questions : 1 How do the colonies on the surface of the pour plate differ differ from the

colonies suspended in the agar ? 2 What is the purpose of inverting the petri dishes in a pour plate technique ? 3 What is the advantage of streak plate over pour plate technique ?

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IX. CULTURAL CHARACTERISTICS OF MICROORGANISMS

Microorganisms when grown on a variety of media will exhibit differences in the macroscopic appearance of growth. These differences are called as cultural characteristics. The growth characteristics are typical of the group or species of most of the bacteria. They are used in the identification and separation of microorganisms into taxonomic groups. These are determined by culturing the organisms in nutrient agar plates, slants and broth. Exercise 11: Growth and cultural characteristics of microbial cultures Aim: To study the growth and cultural characteristics of different organisms

grown in broth or on agar slants and agar plates Materials: 1. Nutrient slants 2. Nutrient broth 3. Nutrient agar plates 4. Broth cultures of different organisms 5. Bunsen burner 6. Inoculating loop and needle Procedure: 1. With a marker pen label the each plate and tube with the name of the organism to be inoculated. 2. Using aseptic techniques, inoculate each of the following media with cultures

Nutrient agar plates: with a sterile loop, prepare a streak-plate inoculation of each of the cultures for the isolation of discrete colonies. Nutrient agar slants: with a sterile needle, make a single line streak of each of the culture. Start at the butt and draw the needle up the center of the slanted agar surface

Nutrient broth: Using a sterile loop, inoculate each organism into a tube of nutrient broth. Shake the loop a few times to dislodge the culture.

3. Incubate all cultures at 370C for 24 to 48 hours. 4. The patterns of the growth to be considered in each of these media are

described below.

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Nutrient agar plates: The well isolated colonies on nutrient agar plates are described as 1. Size: Pinpoint, small, moderate or large 2. Pigmentation : colour of the colony 3. Form: The shape is described as circular, irregular or rhizoid (root like

spreading growth 4. Margin: The appearance of the outer edge of the colony is Entire- sharply

defined, even ; Lobate-marked indentations; Undulate-wavy indentations; Serrate-tooth like appearance; and Filamentous- thread like, spreading edge

5. Elevation : The degree of raise of the colony on the agar surface is described as Flat--elevation not discernable; Raised-- slightly elevated; Convex- Dome shaped elevation; and Umbonate--raised with elevated convex central region

Nutrient agar slants: They have a single line of inoculation on the surface and are evaluated in the following manner: 1. Abundance of growth: The amount of growth is designated as none, slight,

moderate, or large. 2. Pigmentation: Chromogenic microorganisms may produce intracellular

pigments that are responsible for the coloration of the organism as seen in surface colonies. Other organisms produce extracellular soluble pigments that are excreted into the medium and also produce a color. Most organisms, however, are nonchromogenic and will appear white to gray.

3. Consistency: May be evaluated on the basis of the amount of light

transmitted through the growth. This is described as opaque (no light transmission), translucent (partial transmission), or transparent (full transmission).

4. Form: The appearance of the single line streak of growth on the agar surface

is designated as a) Filiform: Continuous, threadlike growth with smooth edges. b) Echinulate: Continuous, threadlike growth with irregular edges. c) Beaded : Nonconfluent to semiconfluent colonies. d) Effuse: Thin, spreading growth. e) Arborescent: Treelike growth. f) Rhizoid: Rootlike growth. Nutrine Broth Cultures: These are evaluated as to the distribution and appearance of the growth as follows:

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1. Uniform fine turbidity: Finely dispersed growth throughout. 2. Flocculant : Flaky aggregates dispersed throughout. 3. Pellicle: Thick, padlike growth on surface. 4. Sediment: Concentration of growth at the bottom of broth culture may be granular, flaky, or flocculent. Questions: 1. Describe the cultural characteristics of Staphylococcus aureus, E. coli and Lactococcus lactis ssp. lactis ?

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X. BIOCHEMICAL REACTIONS Biochemical reactions of microorganisms are very useful in their separation and identification during microbial analysis. These reactions are essentially controlled by enzymatic activity with the help of by biological catalysts called enzymes. They are responsible for bioenergetics, biosynthesis and biodegradation. There are two types of enzymes such as exoenzymes and endoenzymes. Exoenzymes act outside the cell on substances that are difficult to be transported into the cell and degrade these high molecular weight substances into simpler compounds to facilitate transportation through the cell membrane. Example of these reactions are : Lipid hydrolysis, casein hydrolysis, gelatin hydrolysis, starch hydrolysis etc., Endoenzymes function inside the cell and are mainly responsible for the synthesis of new protoplasmic requirements and production of cellular energy from assimilated nutrients. Ex. Carbohydrate fermentation, catalase and oxidase activity, hydrogen sulfide production, litmus milk reactions etc., Exercise 12: Study of biochemical characteristics of bacteria Aim: To perform the biochemical test procedures for identification of bacterial cultures A) Lipid hydrolysis Many microorganisms produce exoenzyme lipase to breakdown the lipids hydrolytically into soluble glycerol and three fatty acids. In the experiment tributyrin is taken as substrate for hydrolytic breakdown. Materials: 1. Tributyrin agar (see annexure) plates 2. Trypticase soy broth (see annexure) cultures of E.coli ,Bacillus cereus and

Pseudomonas aeruginosa. 3. Bunsen burner, 4. inoculating loop. Procedure 1. Prepare the tributyrin agar plate(s) for inoculation as follows:

Divide the bottom of the Petri dish into three sections; lable the first section with E.coli, the second with B.cereus and the third with P. aeruginosa.

2. Using sterile technique, make a single line streak inoculation of each test organism on approximately labeled section of the agar surface.

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3. Inoculate the plate(s) in an inverted position for 24 to 48 hours at 37°C. 4. Examine for the hydrolysis of the tributyrin. A positive test is indicated by a zone of lipolysis which is demonstrated by a clear area surrounding the bacterial growth. B) Casein hydrolysis Casein the milk protein is composed of amino acid sub units linked together by peptide bonds. It is incapable of penetrating the bacterial cell and thus need to be subjected to proteolysis by proteinase enzyme. During proteolysis the casein undergoes stepwise degradation into peptones, polypeptides, dipeptides and ultimately into amino acids and these water soluble amino acids can easily get transported into cell. Materials 1. Milk agar plates 2. Trypticase soy broth (see annexure) cultures of E.coli, Bacillus cereus and

Pseudomonas aeruginosa. 3. Bunsen burner, 4. inoculating loop. Procedure: 1. Prepare the milk agar plate(s) for inoculation as follows: Divide the bottom of the Petri dish into three sections; lable the first section

E.coli, the second B.cereus a and the third P.aeruginosa. 2. Using sterile technique, make a single line streak inoculation of each test

organism on approximately labeled section of the agar surface. 3. Inoculate the plate(s) in an inverted position for 24 to 48 hours at 37oC. 4. Examine for the hydrolysis of the casein. A positive test is indicated by a zone

of proteolysis which is demonstrated by a clear area surrounding the bacterial growth

C) Gelatin hydrolysis Gelatin is a protein produced by the hydrolysis of collagen, a major component of connective tissue and tendons in animals and humans. The gelatin remains in a solid state at below 25°C and in liquid state at above 25°C. Some organisms are capable of producing an exoenzyme called gelatinase which hydrolyses gelatin into amino acids. Upon hydrolysis the gel characteristics of this protein are lost and then it remains liquid even at 4°C.

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Materials 1. Nutrient gelatin deep tubes 2. Trypticase soy broth (see annexure) cultures of E.coli ,Bacillus cereus and

Pseudomonas aeruginosa. 3. Bunsen burner, 4. inoculating loop. Procedure 1. Label each of the nutrient gelatin deep tubes with the name of the bacterial

organism to be inoculated 2. Using the sterile technique, inoculate each experimental organisms into

appropriately labeled deep tube by means of a stab inoculam. 3. Keep one of the nutrient gelatin deep tubes, uninoculated as a control. 4. Incubate all the tubes for 24 to 48 hours at 37° C. 5. The tubes are transferred to refrigerator till the control tube gets solidified.

Compare this control tube with other tubes in test. The tubes with organism that show solidification are negative for gelatinase production and those that show liquification are positive for gelatinase production

D) Litmus milk reactions: Litmus milk forms an excellent differential medium in which microorganisms can metabolize milk substrates depending on the enzyme production. To distinguish among the metabolic changes produced in milk, a pH and an oxidation-reduction indicator litmus is incorporated into the medium. The various biochemical changes that can be observed in milk in this technique are: litmus reduction, curd formation, lactose fermentation, proteolysis, gas production, and alkaline reaction. Lactose fermentation results in the formation of lactic acid which can be detected by the change of colour of litmus from purple (at neutral pH) to pink ( at acid pH of 4.0). The microorganisms produce two different type of curd formations. The first one is formation of acid curd due to the production of lactic acid from the metabolism of lactose. Such clot or curd remains immovable on inverting the tube. Another type of curd is formed by the action of rennin like enzyme on casein and this clot

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or curd is insoluble, soft and semi-solid and also flow out slowly when tube is tilted. Some organisms while fermenting lactose produce gas which can be observed by the separation of the curd or by the development of cracks or fissures within the curd. Some of the organisms that do not utilize lactose will produce enzymes such as proteinases for the purpose of obtaining energy. The action of these enzymes in milk results in proteolysis (Petonization) producing amino acids and ammonia and changing the reaction of the medium into alkaline. In such conditions litmus changes into deep purple colour in upper portion of the milk tube and produces a translucent, brown, wheylike appearnce due to the hydrolysis of protein into amino acids. Materials: 1. Litmus milk broth (see annexure) 2. Bunsen burner 3. Inoculating loop 4. Tripticase soy broth cultures of Escherichia coli, Lactococcus lactis ssp.lactis,

Pseudomonas fluorescences, Enterococcus faecalis. Procedure: 1. Take the litmus milk broth tubes and using sterile loop inoculate the tubes

separately with each of the test organisms and label the tubes appropriately 2. Leave one tube without any inoculation as control 3. Incubate all the tubes at 37°C for 24 to 48 hours. 4. Observe for the changes in litmus milk and note as follows; Sl.No Reaction Change in litmus milk 1 No change Milk remains purplish blue 2 Acid production The milk changes into pink colour 3 Acid with reduction Milk is white with pink band at the surface 4 Litmus reduction Milk remains white with purple band at surface 5 Alkaline reaction Milk will be unchanged with deep blue colur. 6 Acid with reduction and

curd formation Milk becomes curdled and remains white with pink band at the surface

7 Acid with reduction plus gas production

Milk becomes curdled and remains white with pink band at the surface. Fissures in the curd will be observed

8 Proteolysis Milk looks like wheylike brownish translucent medium with deep purple band at the surface

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E) IMViC Tests Differentiation of certain principal group of enterobacteriaceae can be made on the basis of their biochemical properties and enzymatic reactions in the presence of certain specific substrates. The IMViC tests are one of the important group of tests that can be used for this purpose. i) Indole test Certain organisms produce an enzyme known as tryptophanase which hydrolyses the essential amino acid tryptophane. During the reaction indole is produced which is detected by using Kovac's reagent. Materials 1. SIM agar / Casein agar (see annexure) deep tubes 2. Trypticase soy broth cultures of E.coli, Enteobacter aerogenes 3. Bunsen burner, 4. inoculating loop 5. Kovac's reagent Procedure 1. Label each of the SIM deep tubes with the name of the bacterial organism to

be inoculated. 2. Using sterile technique , inoculate each experimental organism into its

appropriately labeled deep tube by means of a stab inoculam. 3. Incubate all of the tubes for 24 to 48 hours at 37°C. 4. Add few drops of Kovac's reagent and observe for colour reaction. Cultures

producing a red reagent layer following addition of Kovac's reagent are positive for indole test.

ii) Methyl red test Glucose is the major carbohydrate oxidized by the enteric organisms for energy production. The end products of this oxidation will vary depending upon the metabolic pathway of the organism. Methyl red is used as pH indicator to detect a large concentration of acid as end product produced by certain organisms. This indicator turns red in the pH range of 4 and yellow in the pH range of 6

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Materials 1. MR-VP broth (see annexure) tubes 2 Trypticase soy broth (see annexure) cultures of E.coli and Enterobacter aerogenes 3. Bunsen burner, 4. Inoculating loop 5. Methyl red reagent (see annexure) Procedure 1. Label each of the MR-VP broth tubes with the name of the bacterial organism

to be inoculated 2. Using sterile technique, inoculate each experimental organism into its

appropriately labeled deep tube by means of a stab inoculam. 3. Incubate all of the tubes for 24 to 48 hours at 37°C. 4. Add a few drops of methyl red indicator and observe for the change in colour. 5. Cultures producing a red reagent layer following addition of methyl red

reagent are positive for methyl red test. iii) Voges-Proskaur test The Voges-proskauer test determines the capability of certain bacteria to utilize the organic acids resulted from glucose metabolism for the production of neutral or non acidic end products like acetylmethylcarbinol. On oxidation this end product acetylmethyl carbinol releases a compound known as diacetyl in the presence of Barritt's reagent producing a deep rose color. Materials 1. MR-VP broth (see annexure) tubes 2 Trypticase soy broth (see annexure) cultures of E.coli ,and Enterobacter

aerogenes 3. Bunsen burner, 4. Inoculating loop 5. Barritt's reagent (see annexure) Procedure 1. Label each of the MR-VP broth tubes with the name of the bacterial organism

to be inoculated

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2. Using sterile technique, inoculate each experimental organism into its

appropriately labeled deep tube by means of a stab inoculation. 3. Incubate all of the tubes for 24 to 48 hours at 37° C. 4. Add a few drops of Barritt's reagent and observe for the change in colour.

Cultures producing a deep rose color reagent layer following addition of Barritt's reagent are positive for Voges-proskauer test

iv) Citrate test Some organisms are capable of utilizing citrate as a carbon source in the absence of glucose or lactose for their energy. This utilization is mediated through an enzyme citrate permease that facilitates the transportation of citrate into the bacterial cell. Citrate inside the cell undergoes enzymatic degradation and finally producing pyruvic acid and carbon dioxide. This carbon dioxide combines with sodium to form sodium carbonate and converting the reaction of the medium into alkaline. Now the bromothymol blue indicator previously incorporated into the medium turns the medium from green into deep Prussian blue. Materials 1. Simmon's citrate agar (see annexure) slants 2 Trypticase soy broth cultures of E.coli, and Enterobacter aerogenes 3. Bunsen burner, 4. Inoculating loop Procedure 1. Label each of the Simmon's citrate agar slants with the name of the bacterial

organism to be inoculated. 2. Using sterile technique, inoculate each experimental organism into its

appropriately labeled deep tube by means of a stab inoculam. 3. Incubate all of the tubes for 24 to 48 hours at 37° C 4. Following the incubation observe the tubes for the change of colour in the

medium. Culture positive for citrate test are identified by the presence of growth on the surface of the slant accompanied by deep Prussian blue color discolouration

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F) Catalase test : Aerobic, microaerophilic and facultatively anaerobic organisms use aerobic respiratory pathway during degradation of carbohydrates for energy production using oxygen as final electron acceptor. During the process they produce hydrogen peroxide which is when accumulated into large concentration, may be toxic to the organisms. Some organisms capable of producing catalase or peroxidase rapidly degrade hydrogen peroxide. Materials: 1. H2O2 solution 2. Cultures of Staphylococcus aureus and Lactococcus lactis ssp. Lactis 3. Nutrient agar slants Procedure:

1. Take the Nutrient agar slants and separately inoculate with Cultures of

Staphylococcus aureus and Lactococcus lactis ssp. lactis 2. Incubate inoculated slants at 37oC for 48 hours 3. Pour 0.5 ml of H2O2 solution over the surface of the each bacterial culture and

observe for the effervescence and note the observation. If catalase production is present bubbles of oxygen are released from the surface of the growth

Questions: 1. Why is gelatin used as solidifying agent instead of agar in gelatin hydrolysis

test ? 2. Differentiate between respiration and fermentation? 3. What is the disadvantage of litmus milk in diagnostic tests? 4. What are the different changes observed in milk when colloidal casein is

hydrolysed? 5. Write the chemical reaction for a positive tributyrin hydrolysis test?

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XI. EFFECT OF PHYSICAL AND CHEMICAL FACTORS ON THE GROWTH OF BACTERIA

Exercise 13: Effect of physical and chemical agents on growth of bacteria Aim: 1. To study the effect of physical factors like temperature, pH, salt and sugar concentration on the growth of bacteria.

2. To evaluate the efficiency of a chemical disinfectant in checking the growth of bacteria using phenol coefficient test

I) Effect of physical factors A) Temperature: Bacteria are capable of growing at a wide range of temperature i.e minus 5oC to 80oC but for their growth each species requires a narrow range of temperature. The range of temperature preferred by bacteria is genetically determined, resulting in production of enzymes with different temperature requirements. The Minimum growth temperature is the lowest temperature at which a species will be able to grow. A species grows faster at its Optimum growth temperature and the highest temperature at which it will be capable of growing is its Maximum growth temperature. Above the maximum temperature most of the organisms die as their cell enzymes are destroyed. Bacteria can be grouped into three major groups depending upon their temperature requirements as : Psychrophiles (growing within temperature range of 0 to 20oC with an optimum temperature of 15oC), Mesophiles (growing within temperature range of 20 to 45oC with an optimum temperature of 37oC) and Thermophiles (growing above 50°C with an optimum temperature of 55oC). Materials: 1. Bunsen burner 2. Inoculating loop 3. Refrigerator 4. Incubators set at different temperatures 5. BOD incubator 6. Bacterial cultures of Pseudomonas fluorescens, Bacillus stearothermophilus,

Micrococcus luteus, E. coli 7. Petri dishes with nutrient agar

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Procedure: 1. Take the petri dish and draw two lines perpendicular to each other to form four

quadrants on the bottom of the dish. 2. Each quadrant of the petri dish will be inoculated with one of the four bacterial

cultures and should be labeled accordingly. 3. Similarly inoculate the other petri dishes with nutrient agar by using different

bacterial cultures. 4. Invert the petri dishes and incubate one petri dish at each of the following

temperatures : 5oC, 15oC, 37oC and 55oC for 24 to 48 hours. 5. Record the results by examining the relative amount of the growth. The

relative growth is rated as No growth : (-) Minimal Growth : (+) Moderate growth : (2+) Heavy growth : (3+) Maximum growth : (4+) B) pH : The pH of the environment greatly influences the growth and survival of the bacteria and most of these differ in their pH requirements. The specific range for bacteria is between 4 and 9 with the optimum pH being 6.5 to 7.5. Metabolic activities of the microorganisms will result in the production of wastes such as acids from carbohydrate degradation and alkali from protein breakdown and this will cause a shift in pH that can be detrimental to the bacterial growth. Materials: 1. Bunsen burner 2. Inoculating loop 3. Bacterial cultures of Staphylococcus aureus, E. coli and a yeast culture of

Sacharomyces sp. 4. Tubes with trypticase soy broth adjusted to pH 3.0, 5.0, 7.0 and 9.0

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Procedure: 1. Take one set of test tubes with trypticase soy broth adjusted to pH 3.0, 5.0,

7.0 and 9.0. Inoculate this set of tubes with a loopful of one of the test organism

2. Repeat the same with another set of tubes using another test organism 3. Once all the test organisms have been inoculated the tubes are incubated at

37oC for 24 to 48 hours. The tubes inoculated with yeast culture are incubated at 25oC for 72 hours.

4. Record the results for each test organism separately 5. Results are expressed as: (+) for presence of growth and (-) for the lack of

the growth. C) Salt and sugar: Salt and sugar will alter the osmotic pressure of the bacterial environment which in turn influences the bacterial growth. Osmotic pressure is the force with which a solvent moves from a solution of lower solute concentration to a solution of higher solute concentration across a semipermeable membrane. A bacterial cell undergoes plasmolysis when the bacteria are exposed to hypertonic environment and in such cases the water leaves the cell and cytoplasmic membrane draws inward away from the cell wall. However the bacteria are capable of growing in hypotonic environment in which the concentration of solutes outside the cell is lower than inside the cell. Facultative halophiles tolerate salt concentrations up to 10% and extreme halophiles require 15 to 20% salt.

Materials: 1. Bunsen burner 2. Inoculating loop 3. Bacterial cultures of Staphylococcus aureus, E. coli and yeast culture of

Sacharomyces sp. 4. Petri dishes with nutrient agar containing 5%, 10% and 15% salt 5. Petri dishes with nutrient agar containing 10%, 25% and 50% sugar

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Procedure: 1. Take one set of petri dishes with nutrient agar containing different salt

concentrations and draw two lines perpendicular to each other to form four quadrants on the bottom of the dish.

2. Each quadrant of the petri dish with one particular concentration of salt will be

inoculated with one of the four bacterial cultures i.e Staphylococcus aureus and repeat the procedure for other quadrants with other test cultures. The plate should be labeled accordingly.

3. Similarly inoculate the other petri dishes with nutrient agar containing other

salt concentrations by using different cultures. 4. Invert the petri dishes and incubate at 37oC for 24 to 48 hours for bacterial

cultures and at 25oC for 72 hours for yeast culture. 5. Repeat the experiment with petri dishes poured with nutrient agar containing

different concentrations of sugar 6. Record the results by examining the relative amount of the growth.The

relative growth is rated as No growth : (-) Minimal Growth : (+) Moderate growth : (2+) Heavy growth : (3+) Maximum growth : (4+) B) chemical agents: Chrmotherapuetic agents are chemical agents which interfere with microbial metabolism there by producing a static effect or cidal effect on the microorganisms. The bacteriostatic agents will cause a temporary inhibition of growth of bacteria while the bacteriocidal agents will cause the death of bacteria. Environmental conditions such as pH, presence of organic matter, time of exposure, concentration of chemical, solubility and toxicity influence the effectiveness of a substance. Phenol coefficient is useful for determining the effectiveness of a chemical substance, as the effectiveness varies among various chemical substances. In phenol coefficient method the effectiveness of a phenolic based chemical is compared with phenol but it is limited to bacteriocidal phenolic compounds and can not be used to evaluate the bacteriostatic compounds.

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Materials: 1. Bunsen burner 2. Inoculation loop 3. Bacterial culture of Staphylococcus aureus or Salmonella typhi 4. Sterile 1 ml pipettes 5. Nutrient broth tubes 6. Test tubes with phenolic dilutions of 1:80, 1:90 and 1:100 7. Test tubes with dilutions of disinfectant under test of 1:100, 1:150, 1:200,

1:250, 1:300, 1:350 and 1: 400 Procedure: 1. Prepare nutrient broth and dispense into test tubes at the rate of 10 ml each

and sterilize and keep aside. 2. Prepare 5ml serial dilutions of phenol and disinfectant under test, in separate

test tubes as shown in materials. 3. A standard quantity of pure culture of Staphylococcus aureus or Salmonella

typhi is added to the first set of test tubes with each of the dilutions of phenol and test disinfectant.

4. At an interval of 5, 10 and 15 min of inoculation, the sterile nutrient broth

tubes are inoculated with a loopful of the contents of each dilution containing organism and phenol or test disinfectant. Label the tubes accordingly indicating the time of exposure, dilution and type of disinfectant.

5. The test tubes with inoculated nutrient broth are incubated at 37oC for 48 hours.

6. After the incubation period examine the tubes for the presence (+) or absence

of (-) growth of the organism and tabulate the results. Note the dilution of test disinfectant and phenol that killed the bacteria in 10 min but not in 5 min

7. Determine the phenol coefficient as follows: Phenol coefficient of disinfectant = Highest dilution of test disinfectant that destroyed microbe in 10 minute but not in

5 minutes/ Highest dilution of phenol that destroyed microbe in 10 minute but not in 5 minutes

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Ex: If 1: 90 dilution of phenol and 1: 250 of test disinfectant are the highest dilutions in which at 5min exposure the growth was noticed and at 10 min exposure the growth was absent phenol coefficient is 2.77 (250 divided by 90). In otherworlds the test disinfectant is 2.77 times as efficient as phenol

Name of the agent Dilution Exposure time 5 min 10 min 15 min

Phenol 1:80 1:90 1:100

Test disinfectant 1:100 1:150 1:200 1:250 1:300 1:350 1:400

. Questions: 1. Explain the effect of temperature on microbial enzymes? 2. What is the principle of gradient plate in assessing the effect of salt or sugar

on microbial growth? What is the difference between the present test procedure employed and gradient plate?

3. What does phenol co-efficient 4 indicate?

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XII. MICROBES IN ENVIRONMENT Exercise 14: Estimation of bacteria and fungi in air

Knowledge of the numbers and types of microorganisms present in the atmosphere inside dairies, cattle byres and food factories, is very important for controlling contamination of milk, milk products and other food materials. In this exercise the student is required to estimate the bacterial content of air in a milking shed and in the bacteriological laboratory. Aim : To enumerate the number of bacteria and yeasts and molds in air Materials: 1. Sterile petri dishes 2. Nutrient agar (see annexure) 3. Potato dextrose agar (see annexure) Procedure 1. Pour two plates with melted nutrient agar and two plates with Potato dextrose

agar. Allow the media to set and harden. 2. Remove the tops from the plates and place one plate of each medium on the

floor of the milking shed and allow them to be exposed for 5 minutes. Immediately replace the tops on the plates.

3. Place one plate of each medium on the laboratory bench, allow the plates to

be exposed for 15 minutes and immediately replace the tops. 4. Incubate the nutrient agar plates at 37 °C for 24 to 48 hours and the Potato

dextrose agar plates at 22 °C for 4 to 5 days. 5. At the end of the incubation period count the number of colonies in each

plate. 6. Prepare smears of typical bacterial colonies in each plate and examine for morphology by Gram's stain. 7. Record your observations and interpret. ________________________________________________________________ Sample Time of exposure Total count Total count per cubic Inference Feet per min ________________________________________________________________ Milk shed Laboratory ________________________________________________________________

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Interpretation: Number of organisms per c. ft. per minute should not exceed one for satisfactory atmosphere Questions: 1. What are the different types of organisms found in air? 2. Why air is not considered as a good medium for the growth of

microorganisms?

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Exercise 15: Estimation of number and type of organisms in soil Soil is an excellent medium for the growth of a variety of microorganisms that include bacteria, actinomycetes, yeasts, and moulds, protozoa and viruses. Soil environment differs from one location to another and from one period of time to another due to variations in moisture, pH, temperature, and organic and inorganic composition of soil. Different media are employed to support the growth these organisms found in soil. Aim: To enumerate the number and types of organisms in a given sample of soil Materials 1. Sterilized petri dishes, pipettes, sample bottles, and spatula. 2. Nutrient agar. 3. Potato dextrose agar (see annexure). 4. Glycerol yeast agar (see annexure). 5. Soil samples. 6. Dilution blanks (99 ml and 9-ml saline). Procedure I) Sampling of the soil:

Remove all the vegetation from the surface of farm soil. Dig with a sterile spatula to a depth of 2 - 3" inside the soil. Transfer about 20 to 25 g of soil into a glass stoppered bottle. Collect samples from two or three different places. Pulverize a small portion of the soil using a sterile pestle and mortar.

II) Plating the samples :

Separate media are used for different organisms: Nutrient agar for bacteria, Potato dextrose agar for yeasts and molds and Glycerol yeast agar for actinomycetes.

a) Make serial dilutions from 1:10 to one : million using 9ml dilution tubes or 99

ml flasks b) Bacteria: Transfer 1 ml of the suspension from 4, 5 and 6 dilutions to petri

dishes and pour melted and cooled nutrient agar. c) Fungi (Yeasts & Moulds) : Similarly plate the sample from dilutions of 1,2 and

3 and pour melted potato dextrose agar. Adjust reaction of the medium using

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about 1 to 1.5 ml of 10% sterile tartaric acid to every 100 ml of potato dextrose agar prior to pouring .

d) Actinomycetes: Transfer 1 ml of the suspension from 3, 4, 5 and 6 dilutions to

petri dishes and pour melted and cooled Glycerol yeast agar. e) Allow the agar plates to solidify and incubate all the plates in an inverted

position at 30 °C for 3 to 5 days. f) At the end of 3 days remove the plates for counting bacterial colonies and

determine the total number of viable bacteria per gram of soil from the average of duplicate plates in any dilution giving colonies between 30 and 300. Similarly the plates poured with acidified potato dextrose agar and glycerol yeast agar are removed at the end of 5 days and the colonies of yeasts and mould and actinomycetes from respective plates are counted. The average number of yeasts and mould and actinomycetes per gram of soil are calculated as in the case of bacterial counts.

Questions: 1. What are the different types of organisms found in soil? 2. Explain the role of microorganisms in soil fertility? 3. Why soil is considered as a good medium for the growth of microorganisms

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Exercise 16: Enumeration of microorganisms in water The availability of potable water is of great importance whether it is for drinking or for use in food and dairy plant. If the water is contaminated with organic matter it serves as an excellent medium for the growth and multiplication of microbes. The presence of non-pathogenic organisms in water is not of major concern, but intestinal contaminants of faecal origin are very much important because they are responsible for infections such as bacillary dysentry, typhoid fever, cholera and paratyphoid fever etc. Analysis of water samples on a routine basis for the presence of these pathogens is very much difficult. Thus the information on microbiological quality of water is obtained by detecting total bacterial content for assessing the general sanitary quality of water and by detecting the indicator organisms for feacal contamination. Aim: 1. To estimate the total number of bacteria in water for assessing the

general quality of water. 2. To estimate the number of coliforms in water by Most Probable Number

(MPN) method A) Estimation of total number of bacteria in water The bacteria in water comprises of feacal and other types. The feacal types grow well at 37oC with in 24 to 48 hours and others grow better at 20 to 22oC in 3 to 5 days. So the information on both types is very important since the ratio between faecal and other types should be low. Generally acceptable ratio between faecal and other types should be 1:10 Material 1. Sample of water for analysis 2. Tryptone glucose agar (see annexure). 3. Dilution blanks (9 ml) 4. Sterilized petri dishes 5. Sterilized pipettes (1ml) Procedure: 1. Shake the water sample thoroughly by moving the bottle up and down 25

times 2. Prepare serial dilutions 1 in 10 and 1in 100 using the dilution blanks. Mix

thoroughly by rotating the tubes between the palms.

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3. Label two sets of petri dishes for separate incubation i.e one set at 37o C and another

at 22oC. 4. Transfer 1 ml of the sample directly from the bottle (Zero dilution) and 1ml

each from 1 in 10 and 1 in 100 dilution into separate petri dishes (in duplicate).

5. Pour molten and cooled agar at 45oC into the plates and mix the agar with

sample by gently rotating the plates without allowing the agar to flow out . 6. When the agar has been set, invert the plates and incubate one series at

37oC for 48 hours and the other at 22oC for 72 hours. 7. At the end of the incubation period count the number of colonies on the plates

containing between 30 to 300 colonies. In case of zero dilution plates, the number of colonies may be counted even if it is less than 30

8. Calculate the number of organisms per ml of water and record your

observation and interpret the results B) Estimation of coliforms in milk by Most Probable number method: The number of coliforms in water is determined by a statistical estimation called the Most Probable Number (MPN) method. The tubes of lactose broth or MacConkey's broth inoculated with samples of water are being tested and a count of the number of tubes showing acid and gas production is then taken and the figure is compared to statistical tables (McCardy table) Materials 1. Sample of tap water 2. MacConkey’s broth tubes containing Andrades’ indicator and Durham tubes (

single as well as double strength) 3. Sterilized 10 ml and 1 ml pipettes 4. 9 ml dilution blank Procedure 1. Transfer one ml portions of the sample into 5 tubes of single strength broth. 2. Prepare 1/10 dilution of the sample and transfer one ml portions of the dilution

sample into 5 tubes of single strength broth.

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3. Transfer 10 ml portions of the sample into 5 tubes containing 10 ml of double strength medium.

4. These three sets of 5 tubes each containing 0.1 ml, 1 ml and 10 ml of the

sample are incubated at 37oC for 24 hours 5. Examine the tubes for production of acid indicated by the development of red

colour and production of gas in Durham tubes. The tubes showing no change should be incubated for another 24 hours

6. Presence of acid and gas in 3 out of 5 tubes in any dilution is considered as

a positive presumptive test for coliform bacteria. 7. Note the highest dilution containing the smallest amount of water in which

positive test is observed. 8. Record your observations and interpret the results Sample No Dilution Presumptive broth tubes MPN / 100 ml Remarks No. of positive tubes ------------------------------ 0.1ml 1ml 10 ml ________________________________________________________________ 1 2 3 4 Questions: 1. Does the number of bacteria determined by the plate count represent all

organisms present in the water sample? 2. What types of bacteria are commonly found in water? 3. What infectious diseases spreads through contaminated water ? 4. Why is E.coli used as an indicator of the sanitary quality of water? 5. Why lactose is used in preference to glucose in presumptive tests/ 6. How do you confirm the presence of E.coli in water? 7. How do the colonies of a) E.coli and b) E.aerogenes appear on Endo agar

and EMB agar? Confirmation test for coliforms: Select the positive tubes and subject them to confirm the previous results. The broth from positive tubes is streaked on two different media namely eosin methylene blue (EMB) and Endo agar.

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Materials required 1. EMB agar or Endo agar (see annexure). 2. Presumptive positive tubes of MacConkey’s broth 3. 24h. old nutrient broth culture of E.coli 4. 24 h. old nutrient broth culture of E.aerogenes Procedure 1. Pour 10 ml to 15 ml melted EMB or Endo agar into petri dish and allow the

media to set 2. Make three sectors on lower dish by making with glass marking pencil by

inverting the petri dish 3. Move the inoculating needle slightly curved so that the streaking should

ensure the presence of well isolated colonies. 4. Streak with the culture of the positive presumptive tubes gently on the agar

surface to avoid tearing of the medium 5. Similarly streak a loopful of the culture of E.coli in second sector 6. Streak the third sector with the culture of E.aerogenes 7. Invert the plates and incubate at 37 C for 24 hours and record your results Typical colonies of coliforms will appear pink with dark center and metallic sheen on EMB agar while on the Endo agar coliform colonies will appear red in color and growth will darken the medium to deep red.

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ANNEXURE

MOST PROBABLE NUMBER (MPN) INDEX TABLE Number of tubes giving positive reaction out of MPN index

per 100 ml Three tubes of 10 ml each

Three tubes of 1 ml each

Three tubes of 0.1 ml each

0 0 0 <3 0 0 1 3 0 1 0 3 1 0 0 4 1 0 1 7 1 1 0 7 1 1 1 11 1 2 0 11 2 0 0 9 2 0 1 14 2 1 0 15 2 1 1 20 2 2 0 21 2 2 1 28 3 0 0 23 3 0 1 39 3 0 2 64 3 1 0 43 3 1 1 75 3 1 2 120 3 2 0 93 3 2 1 150 3 2 2 210 3 3 0 240 3 3 1 460 3 3 2 1100 3 3 3 >2400

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Media 1. Motility test medium Tryptone : 10 g Sodium chloride : 5 g Agra : 5 g Distilled water : 1000 ml Final pH : 7.2 2. Nutrient medium Peptone : 5.0 g Meat extract : 3.0 g Sodium chloride : 5.0 g Agar : 15.0 g Distilled water : 1000 ml Final pH : 7.4 3. MacConkey's medium Peptone : 20 g Lactose : 10 g Sodium chloride : 5 g Sodium taurocholat:e : 5 g Neutral red : 0.03 g Crystal violet : 0.001 g Distilled water : 1000 ml Final pH :7.2 4. SIM agar medium Peptone : 30 g Beef extract : 3 g Ferrous ammonium Sulfate : 0.2 g Sodium thiosulfate : 0.025 g Agar : 3 g Distilled water : 1000 ml Final pH : 7.3

5. Tributyrin agar

Peptone : 5 g Yeast extract : 3 g Tributyrin : 10 ml Agar : 20 g Distilled water : 1000 ml Final pH :

6. Trypticase soy broth

Trypticase : 15 g Phytone : 5 g Sodiumchloride : 5 g Distilled water : 1000ml Final pH : 7.3

7. Milk agar

Skim milk powder : 100 g Peptone : 5 g Agar : 15 g Distilled water : 1000 ml Final pH : 7.2

8. Endo agar

Peptone : 10 g Lactose : 10 g Dipotassium Phosphate : 3 g Sodium sulfate : 2.5 g Basic fuchin : 0.4 g Agar : 15 g

Distilled water : 1000 ml Final pH : 7.5 9. Nutrient gelatin medium Peptone : 5 g Beef extract : 3 g Gelatin : 120 g Distilled water : 1000ml Final pH : 7.2

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10. Simmon's citrate agar Magnesium sulfate : 0.2 g Mono ammonium Phosphate : 1 g Dipotassium

Phosphate : 1 g Sodium chloride : 5 g Sodium citrate : 2 g Bromothymol blue : 0.08 g Agar : 15 g Distilled water : 1000 ml

Final pH : 6.8 10. MR-VP medium

Poly peptone : 7 g Dextrose : 5 g Dipotassium Phosphate : 5 g Distilled water : 1000 ml Final pH : 6.9

11. Glycerol yeast agar

Yeast extract : 2 g Glycerol : 5 ml Dipotassium Phosphate : 1 g Agar : 15 g Distilled water : 1000 ml Final pH : 7.3

12. Casein agar medium

Pancreatic digest of casein : 2 g Sodium chloride : 0.5 g

Distilled water : 1000 ml Final pH : 7.3

13. EMB agar

Peptone : 10g Lactose : 5 g Sucrose : 5 g Dipotassium Phosphate: 2 g Agar : 15 g Eosin Y : 0.4 g Methylene blue : 0.065 g Distilled water : 1000 ml

Final pH : 7.2 14. Litmus milk medium Skim milk powder : 100 g Litmus powder : 0.75 g Distilled water : 1000 ml

Final pH : 6.8

15. Potato dextrose agar Infusion from potatoes : 200 g Dextrose : 20 g Agar : 15 Final pH : 5.6

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Staining solutions and reagents 1. Aqueous methylene blue Saturated solution of methylene blue (1.5%) : 5 ml Distilled water : 95 ml 2. Loeffler's methylene blue

Saturated alcoholic solution Of methylene blue : 30 ml Potassium hydroxide : 1 ml ( 1% aqueous) Distilled water : 99 ml

3. Gram's staining

Crystal violet

a) Solution B: Crystal violet (90 % dye): 2 g

Ethyl alcohol : 20 ml b) Solution B:

Ammonium oxalate : 0.8 g Distilled water : 80 ml Mix reagents A and B Gram's iodine Iodine : 1 g Potassium iodine : 2 g Distilled water : 300 ml Safranin

Safranin O ( 2.5 % solution in ethyl acohol) : 10 ml

Distilled water : 100 ml 4. Acid alcohol

Ethyl alcohol(95%) : 97 ml Con.HCl : 3 ml

5. Carbol fuschin Solution A: Basic fuschin: 0.3 g Ethyl alcohol (95%): 10 ml Solution B: Phenol : 5 g Distilled water : 95 ml Mix A and B solutions

6. Nigrosine solution

Nigrosine (water soluble): 10 g Distilled water : 100 ml Immerse the contents in boiling water for 30 min Formalin : 0.5 ml Filter twice through filter paper

7. Kovac's Reagernt

Pure amyl alcohol or Isoamyl alcohol : 75 ml p-Dimethyl amino benzoldehyde : 5 g Con HCl : 25 ml

8. Methyl red indicator

Methyl red : 0.1 g Ethyl alcohol : 300 ml Distilled water : 200 ml

9. Barritt's Reagent

Solution A : 5% naphthol in absolute alcohol Solution B : 40% potassium hydroxide

10. Andrade's indicator Acid fuchsin (0.5% aqueous): 500 ml

1 N sodium hydroxide : 10 ml