practical microbiology by farman khan

8/8/2019 Practical Microbiology by Farman Khan

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EXPERIMENT NO: 1

TO STUDY THE DIFFERENT PARTS AND WORKING OF MICROSCOPE.

THEORY:

It is an optical instrument consisting of a combination of lenses which help to magnify theimage which is seen through it. In Microbiology, it is used for the study of morphology that is shape andstructure of organism which are microscopic (which are tiny having diameter less than 45um).

PARTS:

(I). MECHANICAL PARTS

(II). MAGNIFYING PARTS(III). ILUMINATING PARTS

(I).MECHANICAL PARTS:

It consists of a stand or base, stage, handle or limb of microscope.y Stand(base):

It forms the base of microscope. In electrical compound microscope, it is rectangular while in case of simple microscope, it is in the form of a horse shoe.

y Limb:

It is that part of the microscope with the help of which we hold the microscope.

y Stage:

It is a type of a plate-form having a hole in it. On stage the slide and cover slip are placed

with the specimen by the help of stage clips. Mechanical stage move by knobs in two

directions that is up and down.

(II). MAGNIFYING PARTS:

It consists of objectives and eye piece. On one side of optical tube, there are objectives and onother side is eye piece. Eye piece may be of magnifying power 5-10X. Objectives are mounted on theother side of optical tube and the portion where objectives are attached is called nose piece. There are 3 or

4 objective lenses having magnifying power of 10X, 40X, 60X and 100X.

(III). ILUMINATING PARTS:

In some microscopes light source is used while in others mirror is used. Mirror which is used, issingle concave mirror. Above the mirror is filter holder which absorbs some constituents of light.

WORKING PRINCIPLE OF A MICROSCOPE

A microscope is a magnifying instrument. The magnified image of the object (specimen) is first produced by a lens close to the object called the objective. This collects light from the specimen and

forms the primary image. A second lens near the eye called the eyepiece enlarges the primary image,

converting it into one that can enter the pupil of the eye. The magnification of the objective multiplied bythat of the eyepiece, gives the total magnification of the image seen in microscopes having a mechanical

tube length (MTL) of 160 mm. The MTL is the distance between the shoulder of an objective and the rimof the eyepiece.

Examples



Objective Eyepiece Total

magnification magnification magnification010X 10X 0 100 diameters

040X 10X 0400 diameters100X 10X 1 000 diameters

Useful magnificationThe objective provides all the detail available in the image. The eyepiece makes the detail large

enough to be seen but provides no information not already present in the primary image formed by theobjective. The magnification of eyepiece used should therefore be adequate to enable the relevant detail in

the primary image to be seen clearly. Increasing further the magnification will reveal no more detail but

only an image that is more highly magnified and increasingly blurred. Not e: The range of total magnifications within which details in the object are seen clearly in the image

(useful magnification) is usually taken as between 500 and 1 000 times the numerical aperture of theobjective (see following text).

Resolving and defining power of an objective

An objective accepts light leaving the specimen over a wide angle and recombines the diverging

rays to form a point-for-point image of the specimen. Objectives of varying magnifications allow aspecimen to be examined in broad detail over a wide area, and in increasing detail over a maller area. This

increase in magnifying power is always linked to an increase in resolving power. The higher the resolving

power of an objective, the closer can be the fine lines or small dots in the specimen which the objectivecan separate in the image. The resolving power of an objective is therefore of great importance. It is

dependent on what is known as the numerical aperture (NA) of the objective. The NA of an objective isan exact figure that has been worked out mathematically from its equivalent focal length and lens

diameter. It is not necessary to know the details of this calculation. Both the NA and magnification of anobjective are usually engraved on it.

The following are the usual NAs of commonly used objectives:

power NA

10X objective 0.2520X objective 0.45

40X objective 0.65100X (oil) objective 1.25

Working of an oil immersion objective

When a beam of light passes from air into glass it is bent and when it passes back from glass toair it is bent back again to its original direction. This has little effect on low power objectives but withhigh power lenses this bending limits not only the amount of light which can enter the lens but also

affects the NA of the objective and consequently its resolving power. The bending effect and its

limitations on the objectivecan be avoided by replacing the air between the specimen and the lens with an oil which has the same

optical properties as glass, i.e. immersion oil. When the correct oil is used, the light passes in a straightline from glass through the oil and back to glass as though it were passing through glass all the way (seeFig.1). By collecting extra oblique light, the oil provides better resolution and a brighter image. Some

50X objectives and all 100X objectives are used immersed in oil.



Illumination system of the microscope

An adequate, well-aligned, and controllable illumination system is required for good microscopy.This can be achieved by using a microscope with built-in illumination. Whenever possible daylight

illumination should be avoided because it is variable, difficult to use, and rarely adequate for oilimmersion work. A substage condenser is used to collect, control, and focus light on the object. It projects

a cone of light matching the NA of the objective, controlled by the iris diaphragm. It also projects an

image of the light source onto the specimen. The light should just fill the field of view of the eyepiece and back lens of the objective uniformly or the image will not be good. It is particularly important to avoidglare and reflections in the microscope and to adjust the condenser aperture correctly for each objectiveand when examining different specimens. To check for glare, the eyepiece can be removed and the inside

of the microscope tube inspected. Glare is present if the inside of the tube is illuminated.

H ow to minimize glare:

Glare in the microscope is caused by any light reaching the eye which does not go to make up the perfect image but interferes with it and the ability of the objective to distinguish detail in a specimen. The

following are the most practical ways of reducing glare in routine work:

a) Position a microscope with built-in illumination in subdued light, not in front of a window. When this

cannot be done, an eyeshade can help to exclude external glare.

b) Avoid using a larger source of illumination than is necessary. If using an adjustable light source, adjust

the light to illuminate no more than the field of view.

c) Reduce condenser glare by reducing the condenser aperture, i.e. adjusting the iris diaphragm, whenusing low power objectives. This will increase contrast but with some loss of resolving power which is

unavoidable.

Some kinds of specimen give more glare than others. A stained blood film or bacterial smear with

no cover glass and examined with the oil immersion objective gives little glare and should therefore beexamined always with the condenser iris wide open. Unstained particles suspended in water or

physiological saline under a cover glass and examined with the 10X objective, give considerable glare.Preparations such as cerebrospinal fluid, urine, or wet unstained faecal preparations require examination

with the condenser iris considerably reduced.Tungsten and halogen illumination: A quartz halogen illumination is preferred to a tungsten

illumination because it gives a consistent bright white illumination. Most modern microscopes with built-

in illumination systems use low wattage halogen lamps for transmitted light microscopy e.g. 6 V 20 W or 6 V 10 W. A tungsten illumination gives a yellow light and its intensity becomes less as the bulb blackens

with age. If a tungsten illumination only is available, a pale blue filter should be inserted in the filter

holder (see following text).

Filters

The following are the main uses of filters in microscopy:To reduce the intensity of light when this is required. A ground glass light diffusing filter is used to

decrease the brilliance of a light source.

a) To increase contrast and resolution. As previously mentioned, a blue daylight filter is commonly used

with an electric µyellow¶ tungsten lamp. This increases resolution. Green filters also increase resolution.

b) To transmit light of a selected wavelength, e.g. an excitation filter used in fluorescence microscopy.



c) To protect the eye from injury caused by ultraviolet light e.g. a barrier filter as used in fluorescence

microscopy.

Aperture iris diaphragm

This device is part of the substage condenser. It serves to control the angle of the cone of light

emerging from the top of the condenser. When adjusted so that the back lens of the objective, as viewedthrough the eyepiece tube, is just filled with light, the full numerical aperture (NA) of the objective is

being utilized. Under these conditions the objective provides maximum resolution, but some glare may be

present, which reduces image contrast. If the aperture iris is adjusted to fill about 75% of the objective'sthe back lens this is reduced and contrast is improved, without significant lose of image detail. Closingthe iris further will increase contrast but some image detail will be lost. A further problem will be the

introduction of details ("artifacts") that are not actually present in the specimen. Therefore, it is veryimport that the iris not be used to control light intensity. (You can simply this process by placing a small

dot of white paint on the iris adjustment lever near the point where it enters the condenser. Now set the

iris for each objective in turn, placing a similar dot on the condenser body inline with the dot on the lever.Lining up the lever dot, with the respective objective mark, will give you the correct iris setting - no need

to remove the eyepiece.)

Body TubeThis part supports the eyepiece and objectives. It is critical that the tube be constructed so that

these optics share a common axis. Most 20th century scopes with body tubes (i.e., not modular) aredesigned for a mechanical tube of either 160 mm, or 170 mm. Mechanical tube length is the distance from

the top of the eyepiece tube to bottom of the Society threaded objective holder.

Coarse Adjustment Knob

As the name suggests, this control (typically a pair, one on each side) moves either the body tube,

or the stage/substage, up or down in a quick manner. This is accomplished by means of a rack and pinion

assembly. The pinion is a toothed wheel (the knobs are attached to either end of the axial) that rides alonga diagonally grooved bar or "rack", attached to the stage or body tube. A good coarse focus control will

provide smooth, back lash free movement, often adequate for initial focusing at magnifications as high as

400x. Examine the workings of this control carefully when evaluating a scope (while observing aspecimen). It should operate smoothly if your viewing experience is to be a pleasurable one. (Ideally, the

rack and pinion surfaces should be completely free of grease when conducting this test.)

Condenser

This probably the most overlooked microscope component. Very few people recognize the

importance of a good quality, properly adjusted condenser, including many professional microscopists. It

is a vital part of the illumination system, and is designed to collect, control and concentrate light from the

lamp onto the specimen. As with objectives, the optical elements can introduce a variety of aberrationswhich are corrected to varying degrees, depending on the type of condenser one is using. Condensers (as

well as filters and objectives) are available to provide specialized, contrast enhancing illumination such asdarkfield, Rheinberg, polarization, differential interference contrast, and phase contrast. If you are using a

basic bright field condenser, with a simple lamp or daylight (cloudy sky - not direct sunlight) ensure that

it is racked all the way up, so that the top element is almost touching the under side of the slide. Do notmove the condenser up and down to adjust light intensity. To ensure maximum contrast and resolutionremove the eyepiece, look into the tube and open the condenser iris until about two thirds of the



objective¶s rear element is filled with light. As with moving the condenser, the iris diaphragm (aperture

iris) should not be used to control field brightness. If the field of view is too bright use a neutral densityfilter, or lamp rheostat to correct this problem. Microscopes with internal or external lamps that include a

field diaphragm and adjustable condensing system can be set up for Köhler illumination. This technique

provides the best possible illumination.

Condenser focus knob

This control is used to precisely adjust the vertical height of the condenser. A good quality

control mechanism will be appreciated once you get into Koehler illumination, or use differentcondensers that need carefully focused.

Draw tube

At one time "all good instruments" had a body tube equipped with an inner sliding draw tube.

This tube enabled users to the adjust the mechanical tube length when certain accessories were screwed

on between the eyepiece and objective, or when using objectives designed for longer mechanical tubelengths. Today's microscopes no longer have or need such a device.

Eyepiece (Ocular)

The optics in this component magnify the "virtual image" formed by the objective. In addition, asthe virtual image cannot be seen directly by the eye (but can be projected onto a sheet of paper), the

eyepiece converts it to a "real image", which the eye can see. Eyepieces are produced in a number of

different designs. For viewing purposes, the Kellner design is preferable. The top element is anachromatic doublet, and it provides a large, flat, well corrected field of view, compared to the basic

Huygenian design (often supplied with 1950's and earlier stands). An additional bonus is the higher eye

point, which makes viewing more pleasurable (very high eye point models are available for spectaclewears). Although some eyepieces are designed to complement a specific series of objectives(compensating eyepieces), for the most part, they are interchangeable among manufactures. The vast

majority are standardized at either 23 mm or 30 mm outside diameter. An eyepiece cannot improve the

inherent resolution of the image formed by an objective, but one of poor design can degrade it.

Eyepiece tube

A fixed tube into which the eyepiece is inserted. For mainstream, "professional" scopes, the

inside diameter is either 23 mm or 30 mm.

Fine focus knob

This control allows for precise focusing of the specimen. Experienced microscopists use thiscontrol far more than the coarse focus control (especially with today's parfocal objectives). It is absolutely

essential that this control work smoothly, with zero rebound effect (e.g. - set the focus and leave for five

minutes, the image should remain razor sharp). Therefore, this control should be checked carefully under viewing conditions, before investing in a microscope. It is VERY expensive to have a fine focus control

repaired, especially on older models.



Filter holder (carrier)

A swing-out circular carrier, or C-shaped frame, attached to the under side of the condenser body.

Filters for reducing light intensity (neutral density), providing near monochromatic light (colour specific -e.g. "daylight"), polarized light or introducing other special lighting characteristics are placed here. The

diameter of holders has not been standardized although most range between 30 and 32 mm. It is possible

to ignore the holder altogether and place the desired filter directly over the light port in stands with built-in lamps (or in front of external lamps for that matter). When black and white photography was all therage (back when the dinosaurs roamed!), skill in the use of filters often determined the quality of the

captured image. However, with today's digital colour imaging, coloured filters are not used that often.

Foot (base)

It rests on the bench top and supports the stage and body of the microscope, and in many casesalso houses the lamp. A well designed base will ensure that the image does not dance about during

focusing, or while manipulating the specimen. There are a vast number of different base designs.

Limb (arm)

The arm is attached to the foot (in scopes without an inclined viewing head by means of an"inclination joint") and supports the body tube. The shape of the arm, and the way in which the body isattached, are often used to illustrate the history of the microscope's development. Today most student and

research stands (older term for a microscope without optics) have very liner, computer designed arms far different in appearance from the one shown in the photograph (which is a classical "Lister limb" - made

about 1930). In such modern stands the "body tube" has been replaced by two removal parts, a viewing

head and an objective changer, with the top end of the arm forming the middle section. This type of arm isvery strong and can better support additional equipment, such as video cameras. (Also, as the stage, notthe arm moves during set-up, there is no longer any concern about additional weight causing the body

tube to drift downward, and out off focus.)

Mirror (or internal lamp)

At one time all stands came with a mirror, even when a base lamp was supplied. Combined withan outboard light source the mirror serves to direct light into the condenser. Except for specializedmirrors, all are second surface mirrors, in other words the silver coating is applied to the back, rather than

front glass surface. In most cases there are two surfaces, a flat, or "plano" surface for directing a parallel

light beam into the condenser, and a curved or concave surface for directly focusing light onto thespecimen with the condenser removed (use with objectives of 10x or less). Always use the flat surface

with a condenser. The silvering must be free of blemishes, if not they can appear as artifacts in the image.To my knowledge it is not economical to have such mirrors resurfaced, and finding replacements may be

next to impossible. In other words, avoid a used scope with a damaged or missing mirror.

Nosepiece (objective changer)

A rotating device to which objectives are attached. Although it seems hard to understand today,such a convenience was not common place until the advent of the 20th century. The quality of objectivechanger is often a good indication of a microscope's overall quality. It should move smoothly, and most

important, should have a distinct click or feel when an objective is properly "seated". Most older nosepieces can accommodate four objectives. However, if you have a choice between a four and five

place unit, take the latter. As your skill improves the fifth spot will prove useful.



Objective

This, together with the condenser, is the microscope! If you have a poor quality objective nothing

you can do will change this. A bit like car horsepower, "what you got is what you got, there ain't nomore". It is much better to start with two good quality objectives then four mediocre ones. While modern,

fixed focus (not infinite focus) objectives have a standardized "adjustment length" (so called "DIN

standard"), and are Society threaded, the degree of optical correction varies. In many cases the barrel of the objective is engraved with information on its optical characteristics. Objective lenses are very tiny andas a result great care is need to form and assemble such lens systems. This generally translates into time,

which in turn translates to cost. Furthermore, it is of paramount importance that manufactures of such

equipment maintain a very high level of quality control. Keep this in mind when shopping for objectives(complete microscopes as well).

An important point to consider when buying older objectives is compatibility. Before DIN was

universally adopted by main stream manufactures objectives were generally shorter, typically having an

adjustment distance of 37 mm (measured from the shoulder of the attached objective to the plane of focusin the specimen). By contrast DIN objectives are much longer, with an adjustment distance of 45 mm.(The longer barrel provides more room for wider lens combinations needed to improve field-of-view, and

field flatness.) So what does all this mean?

Combining long and short barrel objectives destroys one of the unique features of 20th century

microscopes - parfocality. A revolving nosepiece permits rapid changeover between objectives. In practical terms it is essential that the focus of the image be preserved during the change of objectives.

Parfocal objectives allow this to happen (at worse only slight refocusing is required). If you mix DIN andshort barrel objectives you will be constantly refocusing - or you will not be able to focus at all if thestand is designed for 37 mm objectives. Therefore best not to mix short barrel and DIN objectives. As the

difference in length between the two types of objectives is quite significant, they are relatively easy to

distinguish (always measure from the top of the objective shoulder to the surface of the front element). As

a rough rule, a "short barrel" objective will always be less than 37 mm in length, a DIN objective willalways more than 37 mm (10x and higher), powers above 40x almost 45 mm.

Stage clips

These are the basic stage slide holders. Supplied in pairs, they are adequate for general slide

manipulation up to a maximum of 400x (if properly adjusted, which can be tricky). In the hands of a

skilled operator a good pair can serve very well in this magnification range. However, nothing can beat awell made "mechanical stage". (If your scope lack clips elastic bands may do in a pinch.)

Stage

This is the platform or "stage" that supports the specimen (which are typically mounted on glass

slides). To do this job properly it must be perfectly perpendicular to the optical axis, dead flat and of adequate size. A microscope with a dinged or out of line stage should be avoid. As mentioned above

mechanical stages are often supplied. They may be integrated into the stage itself (with the stage deck moving in one axis rather than the slide holder) or they can be attached. Either way they make the life of a

microscopist more enjoyable. However, as movement as well as size are magnified when using a

microscope, they must be well made and in be maintained in top shape. This is another item to bescrutinized under viewing conditions when shopping for a scope. As with the focus controls, movement

must be smooth and backlash free.



EXPERIMENT NO: 2

PREPARATION OF REAGENTS FOR STAINING.Reagents required:

* Crystal violet stain

* Lugol¶s iodine solution* Acetone-alcohol decolourizer * Neutral red (saffranin)

(a).REAGENT-A: Preparation of crystal violet stain:

Ingredients: (for 1 liter)Crystal violet = 20gAmmonium oxalate = 9g

Ethanol (absolute) = 95mlDistilled water = quantity sufficient to make 1000ml or 1liter

Now, (for 100ml)Crystal violet = 2g

Ammonium oxalate = 0.9gEthanol = 9.5ml

Distilled water = quantity sufficient to make 100ml

Procedure:(1). weigh the crystal violet on a piece of clean paper and transfer it to a brown bottle pre-marked to hold

100ml.(2). Then I took the absolute ethanol and mixed until the dye was completely dissolved.

(3). Then weigh ammonium oxalate and dissolve it in 20ml of distilled water in a separate vessel and addto the stain. Then make the volume to 100ml with distilled water mix well.(4). Label the bottle and store it at room temperature.

(5). Filter a small amount of stain into a dropper bottle. Other stain dispensing containers may also be

used for this purpose.(b). REAGENT-B:

Lugol¶s Iodine solution:Ingredients: (for 1 liter)

Potassium iodide = 20gIodine = 10g


Now, (for 100ml)Potassium iodide = 2g

Iodine = 1g


Procedure:(1) First of all weigh KI & transfer it to an amber coloured bottle pre marked to hold 100ml.

(2) Then add about a quarter of volume of water that is about 25ml and mixed until KI wascompletely dissolved.

(3) weigh the iodine and add it to KI solution and mix it until

iodine is dissolved.(4) Adjust the volume to 100ml mark with distilled water and mix

well, label the bottle and mark it toxic.

(c). REAGENT-C:



Acetone-Alcohol decolourizer:

Ingredients: (for 1 liter)Acetone = 500ml

Absolute ethanol = 475mlDistilled water = 25ml

Now, ( for 100ml):

Acetone = 50mlEthanol = 47.5mlWater = 2 .5ml

Procedure:

(1) First of all mix the distilled water with absolute ethanol and transfer

this solution to a screw-cap bottle of 100ml capacity.(2) Then measure the acetone and mix it to the alcohol solution

immediately and mix well.(3) Label the bottle and mark it highly inflammable.

(4) Then transfer a small amount of reagent to a dispensing container thatcan be closed when not in use.

(d). REAGENT-D:

Neutral Red:Ingredients: (for 1 liter)

Neutral red = 1g

Distilled water = quantity sufficient to form 1000ml(for 100ml)

Neutral red = 0.1gDistilled water = quantity sufficient to form 100ml

Procedure:(1) Weigh neutral red on a piece of paper and transfer it to a bottle of

100ml capacity.

(2) Add about a quarter of the volume of water that is 25ml and mixuntil the dye is completely dissolved.

(3) Add the remainder of water and mix well.(4) Label the bottle and store it at room temperature.

(5) Then at last transfer a small amount of stain to a dropper bottle.



EXPERIMENT NO 3

GRAM STAINING TECHNIQUE

APPARATUS:-

Microscope, Slides, Reagents, Cover Slips.

CHEMICALS:-

Crystal violet stain

Lugol¶s stainAcetone-alcohol decolorizer

Neutral red or safraninBacterial colony

STEPS INVOLVED IN STAINING:-

STEP 1:Fix the smear i.e., fixed smear on the slide as following. Smears should be spread evenly covering an area

of about 15±20 mm diameter on a slide. The precautions which should be taken when handling infectious

material. Emulsify a colony in sterile distilled water and make a thin preparation on a slide. When a brothculture, transfer a loopful to a slide and make a thin preparation. Dry the slide by the use of gentle heat.

STEP II:

Cover the smear with crystal violet for 30-60 minutes.

STEP III:

Rapidly wash up with clean water

STEP IV:Tip off the water & cover the smear with logol¶s iodine for 30-60 seconds.

STEP V:Wash up the iodine with clean water.

STEP VI:

Decolorize it with acetone-alcohal water rapidly and wash with clean water.

STEP VIII:

Cover the smear with safranin stain for 2 minutes.

STEP IX:

Wash the stain with clean water.

STEPX:

Whip the back of the slide clean and place of smear air dry.

STEP XI:



Examine the smear microscopically first with 40 X objective to check the staining and to see the

description of material on the slide and with oil immersion objective to report bacterial cell.

Gram positive bacteria:Stain dark purple with crystal violet (or methyl violet) and are not decolorized by acetone or ethanol.

Examples include species of:

S t aphyl ococcu s Act inom ycesS t reptococcu sCl o st ridiumC or ynebact erium

Gram negative bacteria:Stain red because after being stained with crystal violet (or methyl violet) they are decolorized by acetone

or ethanol and take up the red counterstain (e.g. neutral red, safranin, or dilute carbol fuchsin). Examplesinclude species of:

N eisseria Klebsiella Haemo phil u s Br ucella

S almonella Yersinia

S higella ColiformsVibrio

Gram react ionDifferences in Gram reaction between bacteria is thought to be due to differences in the permeability of

the cell wall of Gram positive and Gram negative organisms during the staining process. Followingstaining with a triphenyl methane basic dye such as crystal violet and treatment with iodine, the dye±

iodine complex is easily removed from the more permeable cell wall of Gram negative bacteria but notfrom the less permeable cell wall of Gram positive bacteria. Retention of crystal violet by Gram positive

organisms may also be due in part to the more acidic protoplasm of these organisms binding to the basic

dye (helped by the iodine).

Resul t sGram positive bacteria . . . . . . . . . . . . . Dark purple

Yeast cells . . . . . . . . . . . . . . . . . . . . . . . Dark purpleGram negative bacteria . . . . . . . . . . Pale to dark red

Nuclei of pus cells . . . . . . . . . . . . . . . . . . . . . . . Red

Epithelial cells . . . . . . . . . . . . . . . . . . . . . . . . Pale red



EXPERIMENT NO: 4

STUDY OF THE PROCESS OF STERILIZATION AND ITS VARIOUS METHODS.

INTRODUCTION:Sterilization is the killing or removal of all microorganisms, including bacterial spores, which

are highly resistant. Sterilization is usually carried out by autoclaving which consists of exposure to steamat 121 degree centigrade under a pressure of 15lb/square inch for 15 min.

PHYSICAL METHOD OF STERILIZATION

The physical agents act either by imparting energy in the form of heat or radiation and byremoving organisms through filteration.

1. HEAT:Heat energy can be applied by three ways:

(i). Moist heat sterilization/Autoclaving:

Moist heat sterilization, usually autoclaving is the most frequently used method of sterilization.Because bacterial spores are resistant to boiling, they must be exposed to high temperature, that cannot be

achieved unless the pressure is increased. For this purpose an autoclave chamber is used in which steam at

a pressure of 15lb/square inch, reaches a temperature of 121 deg centigrade and is held for 15-20 min.This kills the highly resistant spores of Clostridium botulinum, the case of botulism, with a margin of

safety. To test the effectiveness of autoclaving process, spore forming organisms, such as members of Clostridium are used.

(ii). Dry heat sterilization:Sterilization by the dry heat, on the other hand requires temperature in the range of 180 deg

centigrade for 20 min. This process is used primarily for glass-wares and is used less frequently than

autoclaving.(iii). Pasteurization:

It is used primarily for milk consisting of heating the milk to 62 deg centigrade for 2 minfollowed by rapid cooling. This is sufficient to kill the vegetative cells of milk-borne pathogens for e.g.

Mycobacterium bovis, Salmonella, Streptococcus, Listeria and Brucella, but not to sterile the milk.2. RADIATION:

The two types of radiation used to kill the microorganisms are ultraviolet light and x-rays. The

greatest antimicrobial activity of UV light occurs at 250-260nm, which is the wavelength region of maximum absorption by puriene and pyrimidine bases of DNA.

X-rays have higher energy and penetrating power than UV radiation and kill mainly by

production of free radicals e.g. production of OH-radicals by the hydrolysis of light. X-rays kill vegetativecells readily but spores are remarkably resistant probably because of their lower water content. X-rays areused in medicine for sterilization of heat sensitive items, such as sutures and surgical gloves, plastic items

such as syringes.

3.FILTERATION: Liquidswhich are destroyed by heat are sterilized by filtration method. The most commonly used filter is

composed of nitrocellulose and has a pore size of 0.22um. but If more than 0.2um then materials cannot pass.

CHEMICAL METHOD OF STERILIZATION

Chemicals vary greatly in their ability to kill microorganisms. Different chemicals are used for sterilization. For e.g. detergents, phenols and alcohols are used to disrupt the cell membrane of microbes.



Chlorine, iodine, heavy metals, hydrogen peroxide, ethylene oxide, acids and alkalis are used

for modification of proteins.Crystal violet and other dyes are used for the modification of nucleic acids.



EXPERIMENT NO 5

AUTOCLAVE

Autoclave is a device in which pressure is used to produce high temperature steam to achieve

sterilization. The temperature of saturated steam at atmospheric pressure is approximately 100 °C. When

water is boiled at an increasing pressure the temperature at which it boils and of the steam it forms, rises,i.e. temperature increases with pressure. At a pressure of 1.1 bar (15 lb/in2, or 15 psi), the temperature of the saturated steam rises to 121 °C. Autoclaving at 121 °C for 15±20 minutes is required for sterilization.Both the temperature and holding time must be correct.

SI unit for pressure:

Most pressure gauges on autoclaves are calibrated in pounds per square inch (lb/in2, or psi) or in bars.The SI unit for pressure force is the pascal (Pa). In SI units, 1 psi is equivalent to 6.9 k Pa and 15 psi to

104 k Pa. Using bars, 1.1 bar is equivalent to 104 kPa or 15 psi.

TemperatureTo obtain the correct temperature for sterilization all the air must first be removed from the autoclave. A

mixture of hot air and steam will not sterilize. It has been estimated that if about 50% of air remains in the

autoclave the temperature will only be 112 °C and heat penetration will be poor. It must also beremembered that at high altitudes, atmospheric pressure is reduced and therefore the pressure required to

achieve 121 °C will need to be increased. At 2 100 m (7 000 feet) a pressure of 18.5 psi is required to

raise the temperature to 121 °C. The pressure should be raised 0.5 psi for every 300 m (1 000 feet) of altitude. Alternatively, a lower temperature and longer sterilizing time can be used (see later text).

Timing

Before commencing timing, sufficient time must first be allowed for the saturated steam to permeate theentire load and for heat transfer to occur. The heat up time will depend on the type of autoclave and items

being sterilized. To prevent accidents and injury it is important to allow sufficient time after sterilization

for the pressure to return to zero and for the load to cool. If the steam discharge tap is opened before the pressure gauge is reading zero, any fluid in the load will boil and bottles may explode. Several hours may

be required for agar to cool to 80 °C for safe handling.

Other sterilizing cyclesAutoclaving at 121 °C for 15±20 minutes is recommended for most laboratory applications. Autoclavesused for sterilizing instruments and packs used in operating theatres, often referred to as clinical

autoclaves, are usually designed to sterilize at 134 °C for 10 minutes or 126 °C for 11 minutes.Autoclaves operating at 121 °C are sometimes referred to as culture media autoclaves.

In laboratories an autoclave is used for:

y Sterilizing reusable syringes, specimen containers,tubes, pipettes, petri dishes, and other articles

of equipment and laboratory- ware that need to be sterile before use and can withstandautoclaving. Items made from glass, metal, and plastic such as nylon, copolymer, or

polypropylene can be autoclaved.y Sterilizing culture media and swabs for use in microbiology work. Most agar and liquid culture

media can be autoclaved. Steaming at 100 °C in an autoclave with the lid left loose is used tosterilize media containing ingredients that could be decomposed or inactivated at temperatures

over 100 °C.

y Decontaminating specimens and other infectious waste prior to disposal.



Using a manually operated autoclave with thermometer and pressure gauge

1. Add the correct amount of water to the autoclave.

2. When loading the autoclave, leave sufficient space between articles for the steam to circulatefreely. Do not allow articles to touch the sides of the chamber or stand in the water. Use a tray or

wire stand in the bottom of the chamber.

3. Secure the lid of the autoclave as instructed by the manufacturer.4. Open the aircock (air outlet) and close the draw-off cock.5. Set the temperature and pressure to the required value.6. Heat the autoclave. If using an electric autoclave, switch on the power to maximum setting. If

using a model without a built-in heater, apply heat from an electric hot plate, gas burner, or

primus stove. When the water begins to boil, air and steam will be expelled through the aircock.7. Allow the correct length of time for all the air to be expelled as instructed by the manufacturer.

8. At the end of when the sterilization is completed the heat is automatically turn off and will coolnaturally. This will usually take almost an hour particularly for agar culture media to cool

sufficiently for safe handling.9. When the thermometer reads below 80 °C and the pressure gauge registers zero, slowly open the

draw-off cock to vent the autoclave. Open the aircock and wait for a few minutes before opening

the lid until the flash indicator stops.

Caut ion: Wear full face and hand protection when opening and unpacking the sterilizer.

A biological indicator is often autoclaved with other materials this indicator commonly consistsof a culture tube containing a sterile ampule of medium and a paper stripped covered with spores of

bacillus stearothermothilus or closstridun. After autoclaving the ampule is aseptically broken and theculture incubating for several days. If the test bacterium does not grow in the medium the sterilization run

has been successful.



EXPERIMENT NO: 6

OBJECT: FORMULATION OF CULTURE MEDIA.

* PREPARATION OF AGAR MEDIA.

Apparatus:Reagent bottles or flasks, distilled water, powdered agar, Petri-dishes, autoclave, cotton

wool and spirit lamp.Procedure:

First take distilled water in a reagent bottle or flask. If flask is used then it should be tightly

closed by cotton wool. Now add in it agar in powder form. Tightly close it and shake well for completedissolution.

Now place the containers containing liquid media in the autoclave. Set the temperature for about 15-20min.The most suitable temperature is 121 deg centigrade. When the temperature reaches to 121 deg

centigrade, media is taken out and placed in flasks.In the same way Petri-dishes are sterilized in an oven for 20 min by setting the temperature up to 180

deg centigrade. When both the media and Petri-dishes are taken out, the media is poured in Petri-dishes. It

is done near the flame of spirit lamp to prevent the entry of microbes present in the environment near theexperimental place.

Now, the Petri-dishes containing media are placed in Laminar flow hood (LFH) to check the sterility

of media. Now this media can be used to grow the microorganisms.Precautions:

(1). After pouring media in flask, it should be closed.(2). Temperature and pressure should be maintained properly.

(3). Pouring should be done near the flame to avoid the entry of microbes in the sterile media.



EXPERIMENT NO 7

MICROBIOLOGY OF AIR

REQUIREMENTS: -

Dehydrated nutrient agar medium, Petri dishes, spirit lamp, flasks.

PROCEDURE: -

Take the required quantity of dehydrated nutrient agar medium in a conical flask. Add the

required quantity of water. Autoclave the medium along with Petri dishes at 121C for 15 minutes. After completion of the sterilization process, transfer it to laminar flow hood (LFH) and allow the medium to

cool down to 50-60 °C. Then pour the medium in the Petri dishes to make a 3-4mm bed of the media in petri dish and allow it to solidify. If any moisture found in the petri dish then remove the cover (lid) of the

Petri dish in the LFH and allow the medium to dry. Expose the Petri dishes at different spots in the laband denote it as A, B, C, D & E. Expose these plates in the air for different period of time, the exposure

time of the plates is as follows.

For a plate A, the exposure time is 5 minutes.

For a plate B, the exposure time is 10 minutes.

For a plate C, the exposure time is 15 minutes.For a plate D, the exposure time is 2 hours.

For plate E placed in LFH unit the exposure time is 4 hours.

After completion of required exposure time for each plate, collect the plate and cover it and then place it in the incubator at a temp of 35 °C. the incubation time is 24-72 hours.



EXPERIMENT NO 8

MICROBIOLOGY OF WATER

TOTAL VIABLE COUNT

REQUIREMENTS:

Culture media plate, flasks, test tubes beakers, micro pipettes

PROCEDURE:-

1. Take 10ml of water sample (tape water) in a sterile screw cap test tube and close it.

2. Take this sample to laminar flow hood (LFH) safely and place it in the test tube rack vertically.

3. Take 7 prepared sterile media plates.4. Now take already prepared nutrient broth, in a flask and add 9 ml of nutrient broth to test

tubes present in the rack.

5. Now take 1 ml from the sample and add it to 9ml of nutrient broth, it will be 10 times diluted.Shake this test tube to make the mixture homogenized and then take 1ml sample from a test

tube no 1 and add it to test tube no 2 this sample will be diluted up to 100 times i.e. 1/100. In

the next step take 1 ml from test tube no 2 and transfer it to test tube no 3 with the help of a pipette. This sample will be diluted up to 1000 times. Transfer 1 ml of solution from test tube

no 3 to test tube no 4 and on up to 100,000 dilutions.6. From each test tube, introduce 1 ml of sample to petri dishes and spread it well on the nutrient

agar media with the help of cotton swab.7. Let these plates to dry in LFH unit. Place the lids on each plate and cover these plates with

aluminum foils. Place these plates in incubator in inverted position at 35 °C.

Note the results after 24 hours and count the colonies. Select those plates which contain 30-200 colonies.

Count the colonies and put the results in following formula,

No of colonies per ml = no. of colonies on the plate X dilution factor

practical microbiology by farman khan

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