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Introduction to Microbiology and

Laboratory Safety

Introduction to Microbiology and

Laboratory Safety

Biosafety

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Media TypesMedia Types

• General Purpose Media

• Enriched Media

• Selective Media

• Differential Media

• General Purpose Media

• Enriched Media

• Selective Media

• Differential Media

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Media TypesMedia Types• General Purpose Media:

• Supports the growth of many microorganisms • i.e. Luria Agar

• Enriched Media:• Has special nutrients to encourage the growth of fastidious

heterotrophs• i.e. Blood Agar

• Selective Media:• Favors the growth of one type of microorganisms and inhibits the

growth of others• Luria + penicillin Agar

• Differential Media:• Distinguishes between different groups of bacteria on the basis of

biochemical characteristics• i.e. Eosin Methylene Blue Agar

• General Purpose Media:• Supports the growth of many microorganisms • i.e. Luria Agar

• Enriched Media:• Has special nutrients to encourage the growth of fastidious

heterotrophs• i.e. Blood Agar

• Selective Media:• Favors the growth of one type of microorganisms and inhibits the

growth of others• Luria + penicillin Agar

• Differential Media:• Distinguishes between different groups of bacteria on the basis of

biochemical characteristics• i.e. Eosin Methylene Blue Agar

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Microbiology Lab Equipment

Microbiology Lab Equipment

• Microscope (with accessories)• inoculation loops• source of flame (Bunsen burner)• Microscope slides and Cover slips• Gram staining kits (can purchase from science supply store)• Petri dishes and proper growth media • incubators • identification kits • autoclave • Clorox bleach, like you buy at the supermarket, diluted to 5-10% is

the best cleaning agent for labs.

• Microscope (with accessories)• inoculation loops• source of flame (Bunsen burner)• Microscope slides and Cover slips• Gram staining kits (can purchase from science supply store)• Petri dishes and proper growth media • incubators • identification kits • autoclave • Clorox bleach, like you buy at the supermarket, diluted to 5-10% is

the best cleaning agent for labs.

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Microorganism Isolation Techniques

Microorganism Isolation Techniques

• Using an Inoculating Loop• Streaking Methods

• Using an Inoculating Loop• Streaking Methods

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How to hold an Inoculating Loop

How to hold an Inoculating Loop

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Streaking and Flaming Procedure

Streaking and Flaming Procedure

• Flame the loop to sterilize it and let cool. • Position the plate so that the spot of inoculum is nearest the hand not

holding the loop (the opposite hand). • Lift the plate lid with the opposite hand; just enough to get the loop inside

and touch the loop to the inoculum spot. It is often helpful to treat the inoculating loop as if it were a pencil - steadying the loop by resting the heel of the hand against the lab bench.

• Move the loop back and forth across the spot and then gradually continue toward the center of the plate as you sweep back and forth. Use a very gentle and even pressure.

• When creating each phase, do not worry about keeping each pass across the plate separate from previous ones.

• When about 30% of the plate has been covered by the first streaking phase, remove the loop and flame sterilize it.

• Repeat the above procedure for the second phase, but this time pick up some inoculum by crossing into the first phase 2-3 times and then not passing into it again (Figure 1-5).

• Repeat as necessary for the third and fourth phases. After streaking the plate, flame sterilize the loop before setting it down.

• Flame the loop to sterilize it and let cool. • Position the plate so that the spot of inoculum is nearest the hand not

holding the loop (the opposite hand). • Lift the plate lid with the opposite hand; just enough to get the loop inside

and touch the loop to the inoculum spot. It is often helpful to treat the inoculating loop as if it were a pencil - steadying the loop by resting the heel of the hand against the lab bench.

• Move the loop back and forth across the spot and then gradually continue toward the center of the plate as you sweep back and forth. Use a very gentle and even pressure.

• When creating each phase, do not worry about keeping each pass across the plate separate from previous ones.

• When about 30% of the plate has been covered by the first streaking phase, remove the loop and flame sterilize it.

• Repeat the above procedure for the second phase, but this time pick up some inoculum by crossing into the first phase 2-3 times and then not passing into it again (Figure 1-5).

• Repeat as necessary for the third and fourth phases. After streaking the plate, flame sterilize the loop before setting it down.

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Triple Streak MethodTriple Streak Method

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Streak Platehttp://www.sumanasinc.com/webcontent/anisamples/microbiology/streakplate.html

Streak Platehttp://www.sumanasinc.com/webcontent/anisamples/microbiology/streakplate.html

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Streak plate method of isolation

Streak plate method of isolation

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Procedure for Making a ‘Smear’

Procedure for Making a ‘Smear’

• Using aseptic technique remove a colony from a plate or cells from your slant. Be carefully to gently touch the surface of your culture with the inoculating loop.

• Make a circular motion in the middle of the circle to spread the cells equally in this region of the slide

• Add a drop of water in the middle • Mix again • Let Air dry • Run the slide through the flame until the slide is warm ( The frosted

side should be down) This fixes the bacteria to the slide • Let the slide cool • Place in the metal tray or in the rack

• Using aseptic technique remove a colony from a plate or cells from your slant. Be carefully to gently touch the surface of your culture with the inoculating loop.

• Make a circular motion in the middle of the circle to spread the cells equally in this region of the slide

• Add a drop of water in the middle • Mix again • Let Air dry • Run the slide through the flame until the slide is warm ( The frosted

side should be down) This fixes the bacteria to the slide • Let the slide cool • Place in the metal tray or in the rack

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Procedure for Transferring

Microorganisms to a Slant

Procedure for Transferring

Microorganisms to a Slant • 1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer

• 2. Using the pinkie finger of your dominant hand twist the red cap from the tube. Hold in your pinkie and do not place it on the counter

• 3. Pass the mouth of the culture tube across the flame • 4. Direct the inoculating needle into the broth. • 5. Flame the mouth of your broth culture tube and replace the cap. Place it in your rack • 6. Pick up the slant in your non dominant hand • 7. Twist off the red cap • 8. Flame the mouth of the slant tube • 9. Direct the inoculating needle into the tube and “ stab” the agar in the base( butt) • 10. Withdraw on the entry line and when you reach the surface make a simple streak along

the face. • 11. Flame the mouth of the tube and replace the cap. • 12. Flame your inoculating needle and replace in your rack.

• 1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer • 2. Using the pinkie finger of your dominant hand twist the red cap from the tube. Hold in your

pinkie and do not place it on the counter • 3. Pass the mouth of the culture tube across the flame • 4. Direct the inoculating needle into the broth. • 5. Flame the mouth of your broth culture tube and replace the cap. Place it in your rack • 6. Pick up the slant in your non dominant hand • 7. Twist off the red cap • 8. Flame the mouth of the slant tube • 9. Direct the inoculating needle into the tube and “ stab” the agar in the base( butt) • 10. Withdraw on the entry line and when you reach the surface make a simple streak along

the face. • 11. Flame the mouth of the tube and replace the cap. • 12. Flame your inoculating needle and replace in your rack.

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Flaming tubesFlaming tubes

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Transferring Microorganisms to Slant Test Tubes

Transferring Microorganisms to Slant Test Tubes

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Streaking a slantStreaking a slant

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Procedure for Transferring Microorganisms to Broth Test

Tubes

Procedure for Transferring Microorganisms to Broth Test

Tubes• Steps for Transfer of Broth to Broth• Hold loop or needle with dominant hand( right ) • Flame the loop • Hold culture tube in left hand • Remove red cap with pinkie of right hand • Flame mouth of culture tube • Place loop into broth( water) • Flame mouth of culture tube and close • Open culture tube with broth( should be labeled) • Dip loop into new broth and mix • Flame mouth of tube and close • Flame loop • Place to the side of your rack

• Steps for Transfer of Broth to Broth• Hold loop or needle with dominant hand( right ) • Flame the loop • Hold culture tube in left hand • Remove red cap with pinkie of right hand • Flame mouth of culture tube • Place loop into broth( water) • Flame mouth of culture tube and close • Open culture tube with broth( should be labeled) • Dip loop into new broth and mix • Flame mouth of tube and close • Flame loop • Place to the side of your rack

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Identifying Bacteria Cultures:

Identifying Bacteria Cultures:

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Colony MorphologyColony Morphology

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Colony MorphologyColony Morphology• Colony morphology• Color• Shape• Margin• Elevation

• Colony morphology• Color• Shape• Margin• Elevation

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Stains and StainingStains and Staining

• Bacteria are slightly negatively charged at pH 7.0 Basic dye stains bacteria

Acidic dye stains background

• Simple stain Aqueous or alcohol solution of single

basic dye

• Bacteria are slightly negatively charged at pH 7.0 Basic dye stains bacteria

Acidic dye stains background

• Simple stain Aqueous or alcohol solution of single

basic dye

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Procedure for Simple Stains

Procedure for Simple Stains

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Differential StainsDifferential Stains• Gram stain

Crystal violet: primary stain

Iodine: mordant Alcohol or acetone-

alcohol: Safranin decolourizer :

counterstain Gram positive: purple Gram negative: pink-red

• Gram stain Crystal violet: primary

stain Iodine: mordant Alcohol or acetone-

alcohol: Safranin decolourizer :

counterstain Gram positive: purple Gram negative: pink-red

Staphylococcus aureusStaphylococcus aureus

Escherichia coliEscherichia coli

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Differential StainsDifferential Stains

• Acid-fast stain Used to detect Mycobacterium species

• Acid-fast stain Used to detect Mycobacterium species

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Procedure for Gram Stain

Procedure for Gram Stain

• All staining work is to be done at the sink • Care should be taken to work directly over the sink • Place 1 drop of crystal violet stain on the smear ( 1 minute) • Rock or roll the slide to cover the area • Use the water bottle to drip water down the slide • Place 1 drop of iodine on the slide ( 1 minute) • Place 1 drop of alcohol on the slide 10 seconds ( KEY – do not leave on longer

than 10 seconds or it will decolorize) • Place 1 drop of saffranin on the slide for 1 minute • Rinse with water from the bottle • Let the slide air dry •

• All staining work is to be done at the sink • Care should be taken to work directly over the sink • Place 1 drop of crystal violet stain on the smear ( 1 minute) • Rock or roll the slide to cover the area • Use the water bottle to drip water down the slide • Place 1 drop of iodine on the slide ( 1 minute) • Place 1 drop of alcohol on the slide 10 seconds ( KEY – do not leave on longer

than 10 seconds or it will decolorize) • Place 1 drop of saffranin on the slide for 1 minute • Rinse with water from the bottle • Let the slide air dry •

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StreptococcusStreptococcus

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Staphylococcus aureusStaphylococcus aureus

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Gram negative bacilliGram negative bacilli

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Safety in the Microbiology Lab

Safety in the Microbiology Lab

An Introduction to Principles and Practices at

Biosafety Levels 1, 2, 3, & 4

An Introduction to Principles and Practices at

Biosafety Levels 1, 2, 3, & 4

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Microorganism Categories

Microorganism Categories

• How are microorganisms categorized? By genetics to show how they are

related By tissues they infect to show how

they cause disease By pathogenicity and communicability

(also known as their BioSafety Level)

• How are microorganisms categorized? By genetics to show how they are

related By tissues they infect to show how

they cause disease By pathogenicity and communicability

(also known as their BioSafety Level)

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Guidelines for Microorganism Use

Guidelines for Microorganism Use

• Besides federal law and regulations other guidelines exist for the use and control of microorganisms: CDC/NIH Biosafety in Microbiological

and Biomedical Laboratories (BMBL) WHO (World Health Organization)

Biosafety Manual USDA (United States Department of

Agriculture) protocols

• Besides federal law and regulations other guidelines exist for the use and control of microorganisms: CDC/NIH Biosafety in Microbiological

and Biomedical Laboratories (BMBL) WHO (World Health Organization)

Biosafety Manual USDA (United States Department of

Agriculture) protocols

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Guidelines for Microorganism Use

Guidelines for Microorganism Use

The microbes are placed into 4 categories called :

Biosafety Levels (BSL 1-4)

The microbes are placed into 4 categories called :

Biosafety Levels (BSL 1-4)

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BSL LabsBSL Labs• Microbiology Laboratories are set

up and maintained to meet a specific containment level. The designated level conveys information about infection potential and engineering controls implemented to protect workers.

• Microbiology Laboratories are set up and maintained to meet a specific containment level. The designated level conveys information about infection potential and engineering controls implemented to protect workers.

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BSL Agents1 Not known to consistently cause disease in healthy

adults

2 Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure

3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

4 Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission

Biosafety Levels for Infectious AgentsBiosafety Levels for Infectious Agents

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BSL Practice

1 Standard Microbiological Practices

2 BSL-1 practice plus: Limited access, Biohazard warning signs, "Sharps" precautions, Biosafety manual defining any needed waste decontamination or medical surveillance policies

3 BSL-2 practice plus: Controlled access, Decontamination of all waste, Decontamination of lab clothing before laundering, Baseline serum antibody analysis

4 BSL-3 practices plus: Clothing change before entering, Shower on exit, All material decontaminated on exit from facility

Recommended Biosafety Level Practices*

Recommended Biosafety Level Practices*

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BSLSafety Equipment (Primary Barriers)

Facilities (Secondary Barriers)

1 None required Open bench top & sink required

2 Primary barriers = Class I or II BioSafety Cabinets; laboratory coats; gloves; face protection as needed

BSL-1 plus: • Autoclave available

3 Primary barriers = Class I or II BioSafety Cabinets; protective lab clothing; gloves; respiratory protection as needed

BSL-2 plus:• Self-closing, double-door access• Exhausted air not recirculated• Negative airflow into laboratory

4 Primary barriers = Class III BioSafety Cabinets or in combination with full-body, air-supplied, positive pressure suit

BSL-3 plus:• Separate building or zone• Dedicated supply and exhaust, vacuum, and decon systems

Engineering Controls by Biosafety Level

Engineering Controls by Biosafety Level

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Safety ResourcesSafety Resources

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Biosafety Level 1 Standard Microbiological

Practices

Biosafety Level 1 Standard Microbiological

Practices

• Restrict or limit access when working

• Prohibit eating, drinking and smoking in the laboratory

• Pipetting by mouth strictly forbidden

• Restrict or limit access when working

• Prohibit eating, drinking and smoking in the laboratory

• Pipetting by mouth strictly forbidden

2.3

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Biosafety Level 1 Standard Microbiological

Practices

Biosafety Level 1 Standard Microbiological

Practices

2.3

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Standard practices also include:

Standard practices also include:

• Keep work areas uncluttered and clean

• No food in lab refrigerator• Minimize splashes and aerosols• Decontaminate work surfaces

daily• Maintain insect & rodent control

program

• Keep work areas uncluttered and clean

• No food in lab refrigerator• Minimize splashes and aerosols• Decontaminate work surfaces

daily• Maintain insect & rodent control

program

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DecontaminationDecontaminationDecontaminationDecontamination

•Sterilization•Disinfection

•Sterilization•Disinfection

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DecontaminationDefinition

DecontaminationDefinition

• SterilizationThe use of a physical or chemical procedure to destroy all microbial life, including large numbers of highly resistant bacterial spores.

• SterilizationThe use of a physical or chemical procedure to destroy all microbial life, including large numbers of highly resistant bacterial spores.

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• DisinfectionThe use of a physical or chemical procedure to virtually eliminate all recognized pathogenic microorganisms but not all microbial forms (bacterial endospores) on inanimate objects.

• DisinfectionThe use of a physical or chemical procedure to virtually eliminate all recognized pathogenic microorganisms but not all microbial forms (bacterial endospores) on inanimate objects.

DisinfectionDefinition

DisinfectionDefinition

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DecontaminationMethods

DecontaminationMethods

• Heat• Chemical • Radiation

• Heat• Chemical • Radiation

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• Types Moist – steam Dry Incineration

*The most effective method of sterilization

• Types Moist – steam Dry Incineration

*The most effective method of sterilization

DecontaminationHeat

DecontaminationHeat

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• Types Liquids, i.e. chlorox, hydrogen peroxide

Gases, i.e. ethylene oxide

• Types Liquids, i.e. chlorox, hydrogen peroxide

Gases, i.e. ethylene oxide

DecontaminationChemical

DecontaminationChemical

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•General Lab Use - Hypochlorite Solutions Large Spills/Large Organic Load

undiluted from bottle Small Spills/Virus Inactivation

10% - 1:9 General Surface Disinfection

1% - 1:99

•General Lab Use - Hypochlorite Solutions Large Spills/Large Organic Load

undiluted from bottle Small Spills/Virus Inactivation

10% - 1:9 General Surface Disinfection

1% - 1:99

DecontaminationChemical

DecontaminationChemical

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In case of a spillIn case of a spill• Wear disposable gloves • Cover large blood spill with paper towels

and soak with 1% (10000 ppm) of household bleach and allow to stand for at least 5 minutes

• Small spill - wipe with paper towel soaked in 1% bleach

• Discard contaminated towels in infective waste containers

• Wipe down the area with clean towels soaked in a same dilution of household bleach

• Wear disposable gloves • Cover large blood spill with paper towels

and soak with 1% (10000 ppm) of household bleach and allow to stand for at least 5 minutes

• Small spill - wipe with paper towel soaked in 1% bleach

• Discard contaminated towels in infective waste containers

• Wipe down the area with clean towels soaked in a same dilution of household bleach

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Aseptic TechniqueAseptic Technique

• First requirement for study of microbes pure cultures, free of other

microbes • Maintain a clean environment; work

close to the flame

• First requirement for study of microbes pure cultures, free of other

microbes • Maintain a clean environment; work

close to the flame

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Thank youThank you