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Introduction to Microbiology and Laboratory Safety

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Title: Introduction to Microbiology and Laboratory Safety


1
Introduction to Microbiology and Laboratory
Safety
Biosafety
2
Media Types
  • General Purpose Media
  • Enriched Media
  • Selective Media
  • Differential Media

3
Media 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

4
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.

5
Microorganism Isolation Techniques
  • Using an Inoculating Loop
  • Streaking Methods

6
How to hold an Inoculating Loop
7
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.

8
Triple Streak Method
9
Streak Platehttp//www.sumanasinc.com/webcontent/
anisamples/microbiology/streakplate.html
10
Streak plate method of isolation
11
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

12
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.

13
Flaming tubes
14
Transferring Microorganisms to Slant Test Tubes
15
Streaking a slant
16
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

17
Identifying Bacteria Cultures
18
Colony Morphology
19
Colony Morphology
  • Colony morphology
  • Color
  • Shape
  • Margin
  • Elevation

20
Stains 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

21
Procedure for Simple Stains
22
Differential Stains
  • Gram stain
  • Crystal violet primary stain
  • Iodine mordant
  • Alcohol or acetone-alcohol
  • Safranin decolourizer counterstain
  • Gram positive purple
  • Gram negative pink-red

Staphylococcus aureus
Escherichia coli
23
Differential Stains
  • Acid-fast stain
  • Used to detect Mycobacterium species

24
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
  •  

25
Streptococcus
26
Staphylococcus aureus
27
Gram negative bacilli
28
Safety in the Microbiology Lab
  • An Introduction to Principles and Practices at
  • Biosafety Levels 1, 2, 3, 4

29
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)

30
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

31
Guidelines for Microorganism Use
  • The microbes are placed into 4 categories called
  • Biosafety Levels (BSL 1-4)

32
BSL 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.

33
Biosafety Levels for Infectious Agents
BSL Agents
1 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
34
Recommended Biosafety Level Practices
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
35
Engineering Controls by Biosafety Level
BSL Safety 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
36
Safety Resources
37
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

2.3
38
Biosafety Level 1 Standard Microbiological
Practices
2.3
39
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

40
Decontamination
  • Sterilization
  • Disinfection

41
DecontaminationDefinition
  • Sterilization
  • The use of a physical or chemical procedure to
    destroy all microbial life, including large
    numbers of highly resistant bacterial spores.

42
DisinfectionDefinition
  • Disinfection
  • The use of a physical or chemical procedure to
    virtually eliminate all recognized pathogenic
    microorganisms but not all microbial forms
    (bacterial endospores) on inanimate objects.

43
DecontaminationMethods
  • Heat
  • Chemical
  • Radiation

44
DecontaminationHeat
  • Types
  • Moist steam
  • Dry
  • Incineration
  • The most effective method of sterilization

45
DecontaminationChemical
  • Types
  • Liquids, i.e. chlorox, hydrogen peroxide
  • Gases, i.e. ethylene oxide

46
DecontaminationChemical
  • General Lab Use - Hypochlorite Solutions
  • Large Spills/Large Organic Load
  • undiluted from bottle
  • Small Spills/Virus Inactivation
  • 10 - 19
  • General Surface Disinfection
  • 1 - 199

47
In 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

48
Aseptic Technique
  • First requirement for study of microbes
  • pure cultures, free of other microbes
  • Maintain a clean environment work close to the
    flame

49
  • Thank you
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