Download Document

Introduction to Microbiology and Laboratory Safety
Biosafety
Introduction to Microbiology and Lab
Safety
Use and Care of Microorganisms
This area of science, may involve many dangers and hazards while
experimenting. It is the sole responsibility of all
teacher(s)/sponsor(s) to teach students proper safety methods and
sterile techniques.
Roles and Responsibilities of Students & Adults
The Instructor is responsible for working with the student to
evaluate any possible risks involved in order to ensure the health and
safety of the student conducting the research and the humans or
animals involved in the study.
Biosafety
Microbiology Techniques
SAFETY










NO FOOD OR DRINKS!
Wash hands thoroughly
Disinfect counters and work area
Tie hair back
Smock, apron, or lab coat optional
Gloves and goggles optional
Closed toed shoes required
Eyewash in middle back sink
Fire blankets in back on shelf by the door
Fire extinguishers in hallway
SAFETY RESOURCES
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
BIOSAFETY LEVEL 1 - STANDARD MICROBIOLOGICAL PRACTICES
BIOSAFETY LEVEL 1 - STANDARD MICROBIOLOGICAL PRACTICES
•
•
•
•
•
•
Always thoroughly wash hands upon entering
and leaving lab
Keep work areas uncluttered and clean
No food in lab refrigerator
Minimize splashes and aerosols
Disinfect work surfaces daily
Maintain insect & rodent control program
DECONTAMINATION
•
•
Sterilization
Disinfection
CHEMICAL DECONTAMINATION
General Lab Use - Hypochlorite Solutions
•Large
Spills/Large Organic Load
•undiluted from bottle
•Small
Spills/Virus Inactivation
•10%
- 1:9
•General
•1%
Surface Disinfection
- 1:99
DISINFECTION
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.
DECONTAMINATION
Sterilization: The use of physical or
chemical procedures that destroy all
microbial life forms, including highly
resistant bacterial endospores.
Autoclave: Pressurized steam at 15 psi
and 121oC for an average of 20 min (10 –
40 min depending on bulk and load)
IN CASE OF A SPILL
•
•
•
•
•
•
Get the instructor, do not pick up glass!
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
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
Autoclave
Clorox bleach, like you buy at the supermarket, diluted to 5-10% or disinfectant
provided in lab.
MICROSCOPY: THE INSTRUMENTS


In a compound microscope
the image from the objective
lens is magnified again by the
ocular lens.
Total magnification =
objective lens  ocular lens
MICROSCOPY
 Lower stage completely
 Rotate nosepiece to 4x objective
 Find area of specimen on slide with naked eye (you may want to





circle the specimen with a sharpie)
Place specimen over circle of light (adjust to area of interest using
stage dials)
Crank stage all the way up using coarse (large knob) focus while
looking in ocular (specimen should come into focus)
Move 4 and 20x objectives out of the way
Place oil directly on slide and rotate objective labeled with “oil, black
line or 40x.”
Use ONLY fine focus (small knob) only to bring into view through
ocular
ASEPTIC TECHNIQUE
•
•
Required for all microbiology preparations to
assure that contaminants are not introduced.
On a personal note, aseptic technique assures
that infectious agents are not spread to you,
fellow students, or the laboratory surfaces.
GENERAL RULES OF MICROBIOLOGY LABORATORY
•
•
•
•
•
•
The inoculating loop is usually used for making transfers of bacterial
cultures (see next few slides for technique).
Allow the loop to cool sufficiently so that any organisms to be tested
will not be killed by the hot wire, but do not allow the loop to
contact anything during the cooling period or contamination will
result.
Learn to remove and replace the caps or lids efficiently without
setting them on the countertop or leaving the cover off too long.
After the transfer is completed the loop must be sterilized again.
Follow the procedure outlined on the following slides to prevent
splattering of infectious materials.
It is probably easier to work while sitting down.
Attention to details and practice will allow you to work both rapidly
and accurately.
How to hold an Inoculating Loop
FLAMING A LOOP
FLAMING A LOOP
FLAMING A LOOP
SMEAR PREPARATION
 Label top side of slide with small piece of masking tape





(or anything that won’t wash off)
Apply a loop full of water
Obtain bacteria specimen aseptically
Mix bacteria and water
ALLOW SMEAR TO COMPLETELY AIR DRY (DO NOT
TRY TO SPEED UP DRYING BY HEATING!)
After slide has completely air-dried then gently heat fix
in flame by hold slide with a clothes pin and quickly
waving bottom side of slide over flame 2 – 3 times.
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
PROCEDURE FOR SIMPLE STAINS
SIMPLE STAIN
 Prepare slide and air dry (circle specimen on





bottom of slide with sharpie to enable finding
specimen when you put it on the scope)
Heat fix
Apply methylene blue
Let stand for 60 sec
Rinse with water
Dry slide
DIFFERENTIAL STAINS
•
Gram stain
• Crystal violet: primary stain
• Iodine: mordant
• Alcohol or acetone-alcohol:
decolorizer
• Safranin: counterstain
• Gram positive: purple
• Gram negative: pink-red
Staphylococcus aureus
Escherichia coli
PROCEDURE FOR GRAM STAIN
•
•
•
•
•
•
•
•
•
•
•
All staining work is to be done over the sink or trough with light even
flow of running water!
Cover specimen on smear with of crystal violet stain (1 minute)
Rock or roll the slide to cover the area
Rinse completely with water using gentle flow from squirt bottle
Cover specimen with iodine on the slide (1 minute)
Rinse with water
Place 1 drop of alcohol on the slide 10 seconds (KEY – do not leave on
longer than 10 seconds or it will over decolorize)
Rinse with water
Cover specimen with saffranin (1 minute )
Rinse with water from the bottle
Let the slide air and/or blot dry
Streptococcus spp.
Staphylococcus aureus
GRAM NEGATIVE BACILLI
CULTIVATION AND ISOLATION OF BACTERIA
•
•
•
•
•
•
•
Diagnostic bacteriology is concerned with the isolation and identification of bacteria in a
specimen from a patient.
These specimens, unless from a normally sterile site of the body, rarely contain a single
bacterial type, but are mixtures of the disease-producing bacteria and the host's normal
or indigenous flora.
Since accurate studies of the biochemical and the antigenic properties of a bacterial
species are possible only through the use of pure cultures, it is necessary to have a
reliable and rapid method that will permit the isolation of possible pathogenic
organisms.
An inoculum from the specimen is streaked on solid agar in a manner, which physically
separates most of the bacterial types, permitting them to form discrete colonies.
This procedure is facilitated whenever possible by the use of either a selective medium
that inhibits the growth of species not sought or by the use of a differential medium,
which imparts a recognizable appearance to the colonies of the type sought.
Upon transfer of this mixed colony to a medium without the inhibitors, both types of
bacteria may grow, and a pure culture will not be obtained.
Consequently, it is often necessary to streak a second plate of the same selective medium
with a colony from the first selective plate in order to obtain a pure culture of the
bacterial species that you are attempting to isolate.
COLONY ISOLATION
•
1.
2.
3.
Four quadrant technique:
Using a sterile loop, streak cultures (liquid broth or
isolated colonies picked from plates) over one-fourth
of the surface of an agar plate. Then flame the loop
again.
Air cool a flamed loop or cool it by touching an
unstreaked area of agar on the same plate.
Pass the cooled loop three or four times over the initial
streaked portion of the plate. Streak it, without
overlap, to the next quadrant.
COLONY ISOLATION
4.
5.
6.
Flame the loop and allow it to cool as
described above in Step 2.
Pass the loop over the streaked portion of
the second quadrant two or three times and
then streak the material without
overlapping over the third quadrant of the
plate.
Repeat Step 5 to streak the last quadrant.
COLONY ISOLATION
STREAK PLATE
STREAK PLATE GOOD AND BAD
PROCEDURE FOR MAKING A SMEAR
1.
2.
3.
4.
5.
6.
7.
Place a very small drop of water on your clean slide.
Using aseptic technique remove a colony from a plate or cells
from your slant. Be careful to just gently touch the surface of
your culture with the inoculating loop.
Transfer inoculum to center of water drop on slide. Mix in a
circular motion to equally spread specimen to the size of
about a dime.
You must allow specimen to then air dry! This may take a
while (5-20 min). You will ruin your smear if you try to
accelerate drying by rapidly heating an in a flame.
Now wave the slide through a flame 3-4 time. This fixes the
bacteria to the slide
Let the slide cool
Place in the metal tray or in the rack
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 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
FLAMING TUBES
TRANSFERRING MICROORGANISMS TO SLANT TEST TUBES
STREAKING A SLANT
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
Flame mouth of culture tube and close
Open culture tube with broth
Dip loop into new broth and mix
Flame mouth of tube and close
Flame loop
Place to the side of your rack
IDENTIFYING BACTERIA CULTURES
COLONY MORPHOLOGY
•
•
•
•
•
Colony morphology
Color
Shape
Margin
Elevation
COLONY MORPHOLOGY
COLONY MORPHOLOGY
Fig. 3.12.a
Fig. 3.12.b
Fig. 3.12.c
Fig. 20.3
Media Types
•
•
•
•
General purpose
Enriched
Selective
Differential
Media Types
•
General purpose:
•
•
•
Enriched
•
•
•
•
fastidious organisms
Streptococcus pyogenes, Neisseria gonorrheae
Blood and chooclate
Selective:
•
•
•
Supports growth of most non fastidious organisms
Nutrient and TSA
Favors the growth of one type of microorganisms and inhibits the growth of
others
Saboraud, EMB, MSA, SS
Differential Media:
•
•
Distinguishes between different groups of bacteria on the basis of biochemical
characteristics
EMB, MacConkey, HE, SS
SAFETY IN THE MICROBIOLOGY LAB
An Introduction to Principles and Practices at
Biosafety Levels 1, 2, 3, & 4
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)
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
GUIDELINES FOR MICROORGANISM USE
Microbes placed in 4 categories:
Biosafety Levels (BSL 1-4)
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.
BIOSAFETY LEVELS FOR INFECTIOUS AGENTS
BSL
Agents
1
Not known to consistently cause disease in healthy adults
(our lab)
2
Associated with human disease, hazard = percutaneous
injury, ingestion, mucous membrane exposure (our lab)
3
Indigenous or exotic agents with potential for aerosol
transmission; disease may have serious or lethal
consequences [Bacillus anthracis (Anthrax)], TB
4
Dangerous/exotic agents which pose high risk of lifethreatening disease, aerosol-transmitted lab infections; or
related agents with unknown risk of transmission (Ebola,
SARS, Swine flu, Hanta virus, West Nile virus)
RECOMMENDED BIOSAFETY LEVEL PRACTICES
BSL
1
2
3
4
Practice
Standard Microbiological Practices
BSL-1 practice plus: limited access, biohazard
warning signs, “sharps" precautions, biosafety
manual defining any needed waste policies for
decontamination or medical surveillance
BSL-2 practice plus: controlled access, decontamination
of all waste, decontamination of lab clothing before
laundering, baseline serum antibody analysis
BSL-3 practices plus: clothing change before entering,
shower on exit, all material decontaminated on exit from
facility
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, mask and eye 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 in combination
with full-body, air-supplied, positive
pressure suit
BSL-3 plus:
• Separate building or zone
• Dedicated exhaust, vacuum, and
decontamination systems