Bacteriology Laboratory

Organization and Skills Dr.T.V.Rao MD Click to edit Master subtitle style

10/14/12

Dr.T.V.Rao MD

11

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 Dr.T.V.Rao MD professionals need

10/14/12

Before staring, be familiar with Normal pathogenic, and opportunistic pathogens

Normal Flora Opportunistic Pathogens Pathogens

Dr.T.V.Rao MD

10/14/12

Microbiology and the Role of the Microbiologists Microbiology study of

microorganisms (simple forms of life visible only with a microscope)

Microorganisms

Normal flora Pathogenic

Microbiology and the Role of the Medical Technicians Medical technicians can be
n

Assists physician / Microbiologists Obtains specimens Prepares specimens for direct examination Prepares specimens for transportation to reference laboratory If office has a POL, performs

Classification by structure

Classification and Naming of Microorganisms

Subcellular DNA or RNA surrounded by a protein coat viruses Prokaryotic simple cell structure with no nucleus or organelles bacteria Eukaryotic complex cell 10/14/12 Dr.T.V.Rao MD 66

Bacteria:

Special groups

Classification and Identification (cont.)

Chlamydia

Mycobacteria bacilli with a cell wall that differs from most bacteria Rickettsia

Cell wall structure differs from other bacteria Live and grow within other living cells

Very small

Live and grow 10/14/12 within other living MD Dr.T.V.Rao

Mycoplasmas completely lack 77 the rigid cell wall

o o o

Single-celled prokaryotic organisms Reproduce rapidly Classification

n

Bacteria

Shape Ability to retain dyes Ability to grow with / without air Biochemical reactions
Dr.T.V.Rao MD 88

10/14/12

Bacteria:

Ability to retain certain dyes

Grams stain Acid-fast stain

Identification (cont.)

Classification and

Ability to grow in presence or absence of air

Aerobes grow best in the presence of oxygen Anaerobes grow best in the 10/14/12 Dr.T.V.Rao MD 99 absence of oxygen

Shape

Classification and Identification

Bacteria:

Coccus spherical, round, or ovoid Bacillus rod-shaped Spirillum spiral-shaped

Dr.T.V.Rao MD 1010

10/14/12

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
Dr.T.V.Rao MD

10/14/12

How Infections Are Diagnosed

o

Steps to diagnosis and treatment

1.

Examine the patient

o Presumptive o May

diagnosis

or may not need additional tests

1. 10/14/12

Obtain specimen(s) Dr.T.V.Rao MD

1212

How Infections Are Diagnosed (cont.)

3.

Examine specimen directly

Wet mount Smear

4.

Culture specimen
n Culture

medium contains

nutrients
n Examine

culture visually and microscopically

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.

Before starting the work ..

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 10/14/12 Dr.T.V.Rao MD refrigerator for later use (2 weeks

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.

Before starting work

Serial dilution and plate count techniques are used to estimate microbial 10/14/12 Dr.T.V.Rao MD

Specimen Collection
o

Must be collected correctly

If not, may not grow in culture n Contaminants may be mistakenly identified n Patient may receive incorrect or harmful 10/14/12 Dr.T.V.Rao MD therapy
n

1616

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

Specimen Collection:
Guidelines
o Avoid causing harm, discomfort, or undue o embarrassment o Collect from appropriate site o Obtain specimen o at correct time 10/14/12 Dr.T.V.Rao MD o Use appropriate o

Obtain sufficient quantity of specimen Obtain specimen prior to the start of antimicrobial therapy Label correctly
1818

Specimen Collection (cont.) Throat culture

specimens
n

Swab back of throat in the area of the tonsils Avoid touching any structures in the mouth Prepare culture plate or prepare correctly

Specimen Collection
o o

(cont.) Urine specimen Sputum specimen

n

n n
n

Clean-catchfrom lungs to minimize Specimen midstream contaminants

Avoid contaminating specimen Process within 60 minutes or with saliva

refrigerate

10/14/12

Dr.T.V.Rao MD

2020

Stool Specimens Wound specimen

n n

Specimen Collection (cont.)

o

Technique varies Swab wound or lesion

o Bacterial infection Do not touch outside of wound

Protozoal or parasitic infection

Instruct patient in correct collection procedure

10/14/12

Dr.T.V.Rao MD

2121

Transporting Specimens to an Outside Laboratory

Many offices send cultures to an outside lab Three main objectives
n

Follow proper collection procedures and proper collection device

2222

Prevent deterioration of specimen Dr.T.V.Rao MD 10/14/12

n

Direct Examination of Specimens

o

Enables physician to initiate treatment immediately Potassium hydroxide (KOH) mounts

Used if a fungal Wet mounts infection of the skin,
n

Nacldissolves keratin that can mask KOH mixed with specimen of fungus slide presence of a glass n Presence of pathogen and movement of microorganism MD 10/14/12 Dr.T.V.Rao 2323
n
n

nails, or hair is suspected

o o

Preparation and Examination of Stained Specimens Grams tentative diagnosis Quick, stain
Moderate- complexity test Differentiation between types of infections Bacteria either retain or lose purple

color

Gram-positive bacteria Gram-negative bacteria

10/14/12

Dr.T.V.Rao MD

2424

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
10/14/12

Mix again

Dr.T.V.Rao MD

2525

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.

10/14/12

Dr.T.V.Rao MD

2626

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.
Dr.T.V.Rao MD 2727 Take the loop and hold it in the

10/14/12

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

10/14/12

Dr.T.V.Rao MD

2828

Media Types
General

Purpose Media Enriched Media Selective Media Differential 10/14/12 Dr.T.V.Rao MD Media

2929

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.

10/14/12

Liquid medium is best when you Dr.T.V.Rao MD 3030 want to rapidly increase the

There are two basic culture techniques used in microbiology:

2.

Culturing Microorganisms

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)

10/14/12

Culture plates are used when you want to test (1) antibiotic sensitivity, (2) estimate culture concentrations from environmental samples, or (3) isolate Dr.T.V.Rao MD 3131 individual colonies from environmental

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

Sterile Technique

When 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 10/14/12 Dr.T.V.Rao MD 3333 sterile techniques.

Organise your Work area

10/14/12

Dr.T.V.Rao MD

3434

Sterile Technique
1.

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) 85% ethanol

2.

10/14/12

Dr.T.V.Rao MD

3535

Aseptic Technique

First requirement for study of microbes

pure cultures, free of other microbes

Maintain a clean environment; work close to the flame

10/14/12

Dr.T.V.Rao MD

3636

Sterile Technique
1.

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)

3737

85% ethanol MD 10/14/12 Dr.T.V.Rao

2.

Culturing Specimens (cont.)

Culture media

Liquid, semisolid, or solid forms Contains agar Selective or nonselective

Dr.T.V.Rao MD 3838

10/14/12

Holding the Inoculating loop

10/14/12

Dr.T.V.Rao MD

3939

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
10/14/12 Dr.T.V.Rao MD 4040

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 10/14/12 in yourDr.T.V.Rao MD 4141 loop live culture. Failure to

Sterility of the Loop Important in Culture Work

10/14/12

Dr.T.V.Rao MD

4242

Inoculating Petri Plates

Step 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 it in 10/14/12 the active microbes and put4343 the Dr.T.V.Rao MD

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
Dr.T.V.Rao MD 4444

10/14/12

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 .

10/14/12

Dr.T.V.Rao MD

4545

Inoculating Petri Plates

Step 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 10/14/12 4646 dish will give Dr.T.V.Rao MD you fewer microbes so

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 10/14/12broth. Dr.T.V.Rao MD 4747 the

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. 10/14/12 Dr.T.V.Rao MD 4848

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
Dr.T.V.Rao MD 4949

10/14/12

Triple Streak Method

10/14/12

Dr.T.V.Rao MD

5050

Optimal results with Scientific Streaking

10/14/12

Dr.T.V.Rao MD

5151

Streak plate method of isolation

10/14/12

Dr.T.V.Rao MD

5252

Colony Morphology

10/14/12

Dr.T.V.Rao MD

5353

Read Colony Morphology

Colony morphology Color Shape Margin Elevation

5454

10/14/12 10/14/12 Dr.T.V.Rao MD Dr.T.V.Rao MD

Determining Antimicrobial Sensitivity

An outside lab reports o Procedure

Sensitive no growth n

Filter paper Intermediate little growth containing Resistant overgrown antimicrobial agents placed on inoculated agar plate
n

Incubated for 24 hours Evaluate 5555

10/14/12

Dr.T.V.Rao MD

Microorganism Categories

How are microorganisms categorized?

By genetics to show how they are related By tissues they infect to show how they cause disease
Dr.T.V.Rao MD 5656

10/14/12

Biosafety is a Concern for all Microbiologists

Click to edit Master subtitle style

10/14/12

Dr.T.V.Rao MD

5757

Restrict or limit access when working

Standard Microbiological Practices

Biosafety Level 1

Prohibit eating, drinking and smoking in the laboratory Pipetting by mouth strictly forbidden
10/14/12 Dr.T.V.Rao MD 5858
2.3

Standard Microbiological Practices

Biosafety Level 1

10/14/12

Dr.T.V.Rao MD

5959

2.3

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 10/14/12 6060 program Dr.T.V.Rao MD

Decontamination

Sterilization Disinfection

Decontamination

Types

Chemical

Liquids, i.e. chlorox, hydrogen peroxide

6262

Gases, i.e. 10/14/12 Dr.T.V.Rao MD ethylene oxide

Decontamination

General Lab Use

Chemical

Hypochlorite Solutions
Large Spills/Large Organic Load

undiluted from bottle 10% - 1:9

Dr.T.V.Rao MD 6363

Small Spills/Virus Inactivation

10/14/12

General Surface Disinfection

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 10/14/12 same dilution of household bleach Dr.T.V.Rao MD 6464

Programme Created by Dr.T.V.Rao MD for Medical Microbiologists in the Developing World

Email

doctortvrao@gmail.com
Dr.T.V.Rao MD 6565

10/14/12