BLS 206: DIAGNOSTIC MICROBIOLOGY

HOZA, A.S (2010)

ROAD MAP…………….

WHAT IS EXPECTED OUT OF THIS COURSE….

AT THE END OF THE COURSE, YOU WILL BE ABLE TO APPLY: MOLECULAR/CONVENTIONAL DIAGNOSTIC MICROBIOLOGY TECHNIQUE IN D’SE Dx!

NB: 1. THERE SHALL BE @ LST 3 EXAMS.

2 . NO FORMAL LECTURE NOTES WILL BE GIVEN

Qn1: Highlight the major steps involved during Gram’s stain in a diagnostic lab

Qn2: Briefly explain why Gram –ve bacteria don’t retain basic stain after decolourization. (Less than 2 lines)

Getting 2 know @other in BLS 206!

Laboratory Diagnosis of Infection

Ask the lab for a diagnosis, expecting a yes or no, but often end up with just a maybe…

Basic Principles of Lab diagnosis

Clinical assessment Collecting and transporting specimens Microscopy Culture Sensitivity Non-cultural diagnostic methods Virological diagnosis

Diagnosis of Bacterial Infection

Patient Clinical diagnosis

HaematologyBiochemistry

Non-microbiological investigations

Radiology

Sample Take the correct specimen

Take the specimen correctly

Label & package the specimen up correctly

Appropriate transport & storage of specimen

A proper clinical assessment is essential for optimal use of laboratory services!

Garbage in

Garbage out

Laboratory

Is your investigation worthwhile?

Do you know whatinformation you want?

Does it affect patientmanagement?

Is the informationalready available?

Contact the lab for info on Best test

Type of sample Timing of sample

Transport of sample Interpretation of results

Give the lab all relevant clinicalinformation

e. g. antibiotic treatment recent travel

special risks etc

stop! thinkagain

yes

no

yes

yes

Can the lab provide thisinformation?

no

no

no

yes

no

no

Happyclinician

Happymicrobiologist

Happypatient

Happymanager

Collecting the correct specimen

Pernasal swabs for pertussis

Sputum , not saliva

Blood culture bottles, not clotted blood

Correctly timed Gentamicin assays

Pus, not swabs

Getting the specimen to the lab Problems in delay or inappropriate storage

delay in diagnosis & treatment pathogens die contaminants overgrow

Blood cultures directly into incubator not refrigerator!

CSF straight to lab

Don't put an entire surgical specimen into formalin! Send a portion to microbiology in a sterile container

Collecting the specimen correctly

Take an mid-stream urineavoids contamination with perineal flora

CSFAvoid contaminationAvoid bloody tap

Throat swabMake the patient gag!

Blood culturesAvoid contamination with skin organisms

Specimens & Infection Control

Please be considerate to lab staff!!

Label hazardous specimens

Don't send specimens to the lab without proper packing

Leaking or blood-stained specimens are not acceptable!!!

Factors limiting usefulness of bacteriological investigations

wrong sample e.g. saliva instead of sputum

delay in transport / inappropriate storage e.g. CSF

overgrowth by contaminants e.g. blood cultures

insufficient sample / sampling error e.g.in mycobacterial disease

Animal/patient has received antibiotics

Diagnosis of Bacterial Infection

culture

on plates or in broth

identification by biochemical or serological tests on pure growth

from single colony

microscopy

Decolorise CounterstainStain

unstained or stained with e.g. Gram stain

sensitivities

Serodiagnosis DNA technologies

by disc diffusion methods,

breakpoints or MICs

Microscopy Unstained preparations

“Wet prep”

Dark-ground illumination for syphilis

Microscopy Stained preparations

Gram-stain Acid-fast stain

Ziehl-Neelsen

Fluorescence Direct, e.g. auramine Immunofluorescence

Culture of Bacteria

Solid media Agar plates

For Identification For Enumeration

Slopes For safe long-term culture,

e.g. Lowenstein-Jensen media for TB

Liquid media (broth) For enrichment or

maximum sensitivity

Advantages of Solid Media

isolation of single clonal colonies get bacterium in pure

culture

identify by colonial morphology

quantification by colony-forming units

Identification of Bacteria

Morphology Growth requirements Biochemistry Enzymes Antigens

Non-cultural diagnostic methods

Antigen detectione.g. latex agglutination

Antibody detectione. g. agglutination tests, complement fixation

tests, indirect immunofluorescence

Molecular methodsPolymerase Chain Reaction

MIC=2mg/L

2mg/L

1mg/L

0.5mg/L

0.25mg/L

4mg/L

8mg/L

amount ofantibiotic

cloudiness meansbacteria can grow atthat concentration of

antibiotic

no zone around disc =resistant

clear zonearound disc =

sensitive

bacterium

Sensitivity tests

on solid media disc diffusion

technique

in liquid media minimum

inhibitory concentration (MIC) test

Breakpoint methods

E-test

I think it’s time for a

short Break!!Break!!

Oooh yeah!!!

Bacterial cultivationPart 1a

Selective /differential

media

Lab activities:

Form 8 groups of about 8 students Each group will have 1 type of specimen

Exercise Demo selective and differentiation plates Streaking bacteria on differentiation plates One group report after each practical MUST

be submitted

Cultivation

The process of growing microorganisms in

culture by taking bacteria from the infection site

(in vivo or environment) and grow them in

artificial environment in the laboratory (in vitro).

Growth needs

Fastidious bacteria – relatively complex growth

needs

Non-fastidious bacteria- relatively basic and

straightforward growth needs

Phases of growth media

Broth:

Growth of bacteria will change the liquid from

clear to turbid (cloudy).

Solid:

Agar plates

Slants

Bacterial cells inoculated on solid media will multiply

enough to be seen by naked eye.

Colony- (clone)

Colony- A bacterial population derived from one bacterial cell. The cells within the colony have identical, genus, species, genetic

and phenotypic characteristics.

Pure bacteria - derived from a single colony.

Selection of a pure colony -most important for bacterial

identification

Media classification and function

Enrichment – Used to enhance growth of a

particular pathogen

Supportive - support growth of most non

fastidious bacteria

Media classification and function

Selective - Contain inhibitory agents that are

inhibitory to all organisms except those sought

Differential - Contain factors that allow bacterial

species to manifest certain metabolic characteristics

that distinguish them from other species.

Media can be both selective and differential based

on the ingredients of the medium.

Blood agar plate (BA)

Nutrient agar with 5% sheep blood

Cultivation of fastidious and non fastidious bacteria.

Differential – Identify hemolysis - Some bacteria secrete

enzymes that lyse red blood cells (hemolysins) such that a

clearing around the colony appears. hemolysis- complete clearing (white hemolysis) hemolysis – incomplete clearing (green hemolysis) hemolysis- no hemolysis

Mannitol Salt Agar (MSA)

Both selective and differential medium.

High salt concentration - inhibits most bacteria.

Selective for Staphylococcus sp.

Differentiate between Staphylococcus sp. by the sugar

mannitol fermentation .

Mannitol fermention produce acids that change the pH

of medium.

Peach color- neutral- no fermentation

Bright yellow- Acidic – mannitol fermentation (Staph. coag.

pos.- Staph. aureus)

MacConkey Agar (MAC)

Selective and differential medium.

Selective - Gram positive bacteria are inhibited by the

presence of bile salts and crystal violet inhibitors in the

medium Most of gram negative bacteria will grow.

Differentiate- Between Gram negative bacteria by their

ability to ferment lactose.

Pink colonies- Bacteria that ferment lactose (precipitation of

some salts in media by acid production).

Pale colonies- Non fermenters

Eosine Methylene blue (EMB)

Differentiatial between

lactose fermenting and

non fermenting enteric

bacteria

Tellurite Glycine Agar (TGA)

Selective- Tellurite

glycine and lithium inhibit

most bacteria

Preferential growth of

Staphyloccoci coagulase

positive (Staphyloccocus

aureus)

Bacteria streaked in lab

Staphylococcus aureus Staphylococcus epidermidis Salmonella pullorum, E.coli

EMB and TGA

S.aureus

S.pullurum

S.epidermidis

E.coli

OK guys, it’s the end of presentation, C U next time...!!