Bacteriology Laboratory Organization

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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

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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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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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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

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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

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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.

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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

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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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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

http://upload.wikimedia.org/wikipedia/commons/5/56/Agarplate_redbloodcells_edit.jpg

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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.

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• 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 .

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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.

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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

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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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• 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

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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

• [email protected]

Health & Medicine

Bacteriology Laboratory Organization