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Bacteriology Laboratory Organization
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04/11/2023 Dr.T.V.Rao MD 1
Bacteriology LaboratoryOrganization and Skills
Dr.T.V.Rao MD
Bacteriology Laboratory
Bacteriology Laboratory makes
the Backbone of any Hospital and without
which no hospital can function to the Minimal needs, All the Microbiologists
and Lab professionals need the basic skills and safety for effective
functioning of services
Before staring, be familiar with Normal pathogenic, and opportunistic
pathogens
• Normal Flora
• Opportunistic Pathogens
• Pathogens
Microbiology and the Role of the Microbiologists
• Microbiology – study of microorganisms (simple forms of life visible only with a microscope)
• Microorganisms–Normal flora–Pathogenic
Medical technicians can be Assists physician / MicrobiologistsObtains specimensPrepares specimens for direct examinationPrepares specimens for transportation to
reference laboratoryIf office has a POL, performs microbiologic
procedures
Microbiology and the Role of the Medical Technicians
04/11/2023 Dr.T.V.Rao MD 6
Classification and Naming of Microorganisms
• Classification by structure–Subcellular – DNA or RNA surrounded
by a protein coat – viruses–Prokaryotic – simple cell structure with
no nucleus or organelles – bacteria–Eukaryotic – complex cell structure with
nucleus and specialized organelles – protozoans, fungi, parasites
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• Special groups– Mycobacteria – bacilli
with a cell wall that differs from most bacteria
– Rickettsia • Very small • Live and grow within
other living organisms such as mites and ticks
– Chlamydia • Cell wall structure
differs from other bacteria
• Live and grow within other living cells
– Mycoplasmas – completely lack the rigid cell wall
Bacteria: Classification and Identification (cont.)
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BacteriaSingle-celled prokaryotic organismsReproduce rapidlyClassification
ShapeAbility to retain dyesAbility to grow
with / without airBiochemical reactions
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• Ability to retain certain dyes – Gram’s stain– Acid-fast stain
• Ability to grow in presence or absence of air– Aerobes – grow best in the presence of oxygen– Anaerobes – grow best in the absence of oxygen
• Biochemical reactions
Bacteria: Classification and Identification (cont.)
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Bacteria: Classification and Identification
• Shape– Coccus – spherical, round, or ovoid
– Bacillus – rod-shaped
– Spirillum – spiral-shaped
– Vibrio – comma-shaped
All Microbiologists should be familiar with :
• Clinically significant bacteria– Morphological characteristics – Biochemical characteristics– Signs and symptoms they cause in the host
they are infecting– Virulence factors– Pathophysiology of infection
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How Infections Are Diagnosed
Steps to diagnosis and treatment1. Examine the patient
Presumptive diagnosisMay or may not need additional tests
2. Obtain specimen(s)Label properly Include presumptive diagnosis
How Infections Are Diagnosed (cont.)
3. Examine specimen directly• Wet mount• Smear
4. Culture specimenCulture medium – contains nutrientsExamine culture visually and
microscopically
Before starting the work ..Different media are used to culture microorganisms, be certain that you are using the appropriate media for your organism.Always use sterile technique to prevent contamination.Choose the type of media (liquid or plate) appropriate for your investigation or application.Sterile liquid culture tubes and media plates can be prepared in advance and stored in the refrigerator for later use (2 weeks for liquid culture tubes, 2 months for media plates).
Before starting work …Liquid culture tubes, solid slant tubes, and petri plates can be used to culture microbes.Media and lab materials should be sterilized prior to use; an autoclave or a pressure cooker can be used in the sterilization process.
Serial dilution and plate count techniques are used to estimate microbial populations from environmental or commercial cultures.
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Specimen CollectionMust be collected
correctly If not, may not grow in
cultureContaminants may be
mistakenly identifiedPatient may receive
incorrect or harmful therapy
Specimen Collection (cont.)
• Devices– Use appropriate collection
device or specimen container– Sterile swabs – absorbent
material on the tip
• Collection and transporting systems– Sterile, self-contained– Transport medium– Aerobic or anaerobic
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Specimen Collection: Guidelines
Avoid causing harm, discomfort, or undue embarrassment
Collect from appropriate site
Obtain specimen at correct time
Use appropriate devices
Obtain sufficient quantity of specimen
Obtain specimen prior to the start of antimicrobial therapy
Label correctly
Specimen Collection (cont.)Throat culture specimens
Swab back of throat in the area of the tonsils
Avoid touching any structures in the mouth
Prepare culture plate or prepare correctly for transport to laboratory
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Specimen Collection (cont.)
Urine specimenClean-catch midstream to minimize
contaminantsProcess within 60 minutes or refrigerate
Sputum specimenSpecimen from lungs Avoid contaminating specimen with
saliva
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Specimen Collection (cont.)
Wound specimenSwab wound or lesionDo not touch outside of wound
Stool SpecimensTechnique varies
Bacterial infectionProtozoal or parasitic infection
Instruct patient in correct collection procedure
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Transporting Specimens to an Outside Laboratory
Many offices send cultures to an outside lab
Three main objectives Follow proper collection
procedures and proper collection device
Prevent deterioration of specimen
Protect anyone handling specimen
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Direct Examination of Specimens
Enables physician to initiate treatment immediately
Wet mountsNacl mixed with
specimen of glass slidePresence of pathogen
and movement of microorganism
Potassium hydroxide (KOH) mounts Used if a fungal infection of the skin, nails, or hair
is suspectedKOH dissolves keratin that can mask presence of a
fungus
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Preparation and Examination of Stained Specimens
Quick, tentative diagnosis Differentiation between types of infections• Gram’s stain
– Moderate- complexity test– Bacteria either retain or lose purple color
• Gram-positive bacteria • Gram-negative bacteria
04/11/2023 Dr.T.V.Rao MD 25
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
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Making a Smear• Wash the glass slide thoroughly
with soap and water then rinse with 95% alcohol to sterilize.
• 2. Allow the slide to dry properly.• 3. Pass the clean slide over a
flame with its face down to further sterilize it. (Make sure to hold it by its edge)
• 4. Draw a small circle on the slide so you can put your bacteria on the back of the marked area.
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Smear Preparation• Smear Preparation• Only a small amount of bacterial
culture should be used.• Thick smear causes overcrowding
of a large number of cells.• Two different media require two
different techniques• Liquid Medium/ Broth Culture• 1. Take the loop and hold it in
the flame at 45o until it turns red. Your loop is inoculated now. Let it cool for a few minutes.
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Procedure for Making a ‘Smear’• 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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Media Types• General
Purpose Media• Enriched
Media• Selective
Media• Differential
Media
• General Purpose Media
• Enriched Media
• Selective Media
• Differential Media
04/11/2023 Dr.T.V.Rao MD 30
Culturing Microorganisms
• There are two basic culture techniques used in microbiology:
1. Liquid culture: bacteria, algae, and some fungi can be reared in culture tubes (test tubes) in a liquid medium. Liquid medium is best when you want to
rapidly increase the concentration of the organism or when you want to grow motile cells.
04/11/2023 Dr.T.V.Rao MD 31
Culturing Microorganisms• There are two basic culture techniques
used in microbiology:2. Culture Plates: Liquid medium is solidified
using agar (Agarose) and poured as a thin layer in the bottom of a culture dish (also sometimes called petri plate) Culture plates are used when you want to test (1)
antibiotic sensitivity, (2) estimate culture concentrations from environmental samples, or (3) isolate individual colonies from environmental samples.
Culturing Specimens in the Laboratory
• More common to send specimens for culture to outside labs
• Culturing involves placing a sample of specimen on a culture medium– Medium – nutrients– Place in incubator for growth – colony develops as
microorganism multiplies
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Sterile TechniqueWhen culturing bacteria or other
microorganisms, it is important to keep your work area as clean as possible.
This prevents the introduction of other microorganisms from the environment into your culture.
The techniques used to prevent contamination are referred to as sterile techniques.
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Organise your Work area
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Sterile Technique1. Start by washing your
down your work or lab benches with a surface disinfectant. The most commonly used disinfectants for lab use are:
1. 10% bleach (recommended by the CDC)
2. 85% ethanol
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Aseptic Technique
• First requirement for study of microbes
–pure cultures, free of other microbes
• Maintain a clean environment; work close to the flame
04/11/2023 Dr.T.V.Rao MD 37
Sterile Technique1. Start by washing your down your
work or lab benches with a surface disinfectant. The most commonly used disinfectants for lab use are:
1. 10% bleach (recommended by the CDC)
2. 85% ethanol
04/11/2023 Dr.T.V.Rao MD 38
Culturing Specimens (cont.)
• Culture media – Liquid, semisolid, or
solid forms– Contains agar– Selective or nonselective
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Holding the Inoculating loop
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Media Types• General Purpose Media:
• Supports the growth of many microorganisms • i.e. Nutrient 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. Nutrient 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
04/11/2023 Dr.T.V.Rao MD 41
Inoculation of Culture Plates and Tubes
Clean and surface sterilize your work area as detailed in the section on Sterile Technique.
Use either disposable inoculation loops or a metal loop that can be heat sterilized to inoculate plates, slants, and liquid culture tubes.
If using a metal loop, be sure to cool the loop by touching the sterile cooled liquid media or the sterile culture plate before the placing the loop in your live culture. Failure to cool the loop will kill your active microbial cultures!
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Sterility of the Loop Important in Culture Work
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Inoculating Petri PlatesStep 1:Remove the culture tube stopper or cap with
one (do not set it down) and flame the mouth of the tube to surface sterilize the mouth. The heated tube surface will generate a thermal current that prevents contamination of the culture.
Step 2: Without setting any of the culture materials on the bench, place the sterile inoculation loop in the culture.
Step 3: Replace cap on the culture tube with the active microbes and put it in the test tube rack.
04/11/2023 Dr.T.V.Rao MD 44
Culturing Specimens (cont.)
• Inoculating a culture plate– Transfer some of the specimen onto a culture
plate– Label the plate correctly– Qualitative analysis – determination of type of
pathogen– Quantitative analysis – number of bacteria
present in sample
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Inoculating Petri Plates
Step 4: Holding the petri dish lid at an 30-45° angle, work the inoculating loop from the outside of the plate toward the center in a zig-zag pattern that covers approximately 25% of the plate surface .
04/11/2023 Dr.T.V.Rao MD 46
Inoculating Petri PlatesStep 5: Turn the petri plate 90° to the right,
dragging the inoculation loop through the last section of the plate, moving from the outside to the inside in a zig-zag motion.
Step 6: Repeat this process twice more until the entire plate surface is covered.
NOTE: If you are trying to isolate individual colonies, each turn of the dish will give you fewer microbes so that you can distinguish individual colonies.
04/11/2023 Dr.T.V.Rao MD 47
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
04/11/2023 Dr.T.V.Rao MD 48
Procedure for Transferring Microorganisms to a Slant
• 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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Culturing Specimens (cont.)
• Inoculating a culture plate– Transfer some of the specimen onto a culture
plate– Label the plate correctly– Qualitative analysis – determination of type of
pathogen– Quantitative analysis – number of bacteria
present in sample
Triple Streak Method
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Optimal results with Scientific Streaking
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Streak plate method of isolation
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Colony Morphology
Read Colony Morphology
• Colony morphology
• Color• Shape• Margin• Elevation
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Determining Antimicrobial Sensitivity
• An outside lab reports– Sensitive – no growth– Intermediate – little growth– Resistant – overgrown
Procedure Filter paper containing
antimicrobial agents placed on inoculated agar plate
Incubated for 24 hoursEvaluate effectiveness of
agent
04/11/2023 Dr.T.V.Rao MD 56
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)
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Biosafety is a Concern for all Microbiologists
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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
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Biosafety Level 1 Standard Microbiological Practices
2.3
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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
Decontamination•Sterilization
•Disinfection
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• Types–Liquids, i.e. chlorox, hydrogen peroxide
–Gases, i.e. ethylene oxide
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
DecontaminationChemical
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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
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• Programme Created by Dr.T.V.Rao MD for Medical Microbiologists in the
Developing World• Email