PowerPoint Sunusu

Rules in the microbiologylaboratory

Rules• Loops and the other metal stuff shouldbe sterilized before and after use.• Liquid media should be kept in tubeholders.• If the working area is contaminated itshould be cleaned with alcohol.• Benches, microscopes and otherlabware used should be cleaned up.• Hands should be washed with soapbefore leaving the laboratory.

Rules• It is recommended to write and drawa report of the lab studies at the endof the laboratory hour.

Inoculation and Evaluation ofClinical samplesFirst Day

ClinicalsampleWrongmediainoculationLaboratoryerrorsFalseresultRepeatof testIrrationalantibioticuse

ContainersFalcon tubeSputum,urine...Sterile containerurine,sputum...Non-sterilecontainersstool...

Transport MediumReady for useUsed

AnaerobicTransport Mediumtablet liquid cotton cotton liquid cotton swab

Microbiological Examination• Macroscopic examination• Microscopic examination• Culture– Selection of media– Inoculation– Incubation– Evaluation (with direct smear)• Macroscopic examination of the colonies• Microscopic examination of the colonies• Manual tests• Automated or semi-automated identification systems• Antibiotic susceptibility testing• Report

Macroscopic examination• Urine– colour– turbidity• Stool– blood, mucus– watery stool• Pus– blood• Throat– .• Sputum– blood, mucus, purulent material

Microscopic examination• Preparation of direct smear from thesample– Wet mount• Examination without staining• Staining with iodine• Staining with indian ink• …– Fixed• Gram staining• Methylene blue staining• Giemsa staining• …

Culture– Selection of medium– Inoculation– Incubation– Evaluation ( with a direct smear ifexists)• Macroscopic examination of the colonies• Microscopic examination of the colonies• Manual tests• Automated or semi-automated identificationsystems• Antibiotic susceptibility testing

Selection of mediaAppropriate media are selected according tothe clinical sample.• Nutrient medium• Enrichment media: Blood,chocolate agar• Selective media: MacConkey, EMB agar• Differential media:– Blood agar: Hemolysis– MacConkey agar,EMB agar: Lactose utilization– SS agar: Lactose utilization, H 2 S production

Selection of the media accordingto the sample• Urine: Blood agar, EMB agar• Throat: Blood agar• Sputum , BAL, DTA: Blood agar, EMB agar, chocolate agar• Stool: EMB agar, SS agar• Blood: Blood culture bottle• CSF: Blood agar, EMB agar, chocolate agar• Sterile body fluids: Blood agar, EMB agar• Pus, wound: Blood agar, EMB agar• Tissue, surgery material: Blood/EMB agar, sampleinoculation into the liquid media• Vagina: Blood agar, EMB agar, chocolate agar, antiboticcontaining Columbia agar• Urethral swab: sheep blood/EMB agar, chocolate agar

Selection of the media accordingto the culture demand• Anaerobe culture: Schaedler, phenyl ethanol, BBE agar,thioglycolate broth, also aerobic culture with blood, EMBand chocolate agars.• Tuberculosis culture: Loewenstein-Jensen medium,Middlebrook medium• Diphtheria : Loeffler medium, blood agar with tellurite• Pertussis: Bordet-Gengou medium• Cholera: TCBS agar, alkaline pepton water• EHEC: Sorbitol containing MacConkey agar• Yersinia: CIN agar• Helicobacter• Campylobacter• Vancomycin resistant enterococcus• ………….

Enrichment media• Some extra nutritional materials canbe added such as blood, egg, serum.• Most of the microorganisms grow onthese media– blood agar– chocolate agar

Selective media• These media are selective because they inhibitsome bacteria and permit the growth of others.- EMB or MacConkey agar– Antibiotic containing chocolate agar (Thayer-Martin agar)

Differential Media• Because of the indicators they have,similar bacteria form different colonies.– Blood agar– EMB or MacConkey agar

• Liquid specimen– Quadrant streak:• Stool• Sputum– Quantitative• Urine• BAL, DTA• Catheter• Swab– Quadrant streak:• Pus• Throat• TissueInoculation

• Purecoloniesfrom allthebacteriaculturedmay beobtained.Quadrant streak platetechnique

Quantitative inoculation• A certain amount ofliquid specimen isinoculated.– 10 µL for urine (aloopful)• Afterwards growthof colonies arecounted to obtainnumber of bacteriaper mL.

Urine• Medium: Blood agar, MacConkey agar• Inoculation:– Quantitative inoculation to blood agar : 10μL (a loopful)– Inoculation with Quadrant streak plate technique toMacConkey agar : 10μL (a loopful)• Incubation: Normal atmosphere, 36-/+2ºC,overnight

Stool• Medium: MacConkey agar, SS agar• Inoculation: Quadrant streak plate technique• Incubation: Normal atmosphere, 36-/+2ºC,overnight

Pus, wound, tissue, drainage andsurgical materials• Medium: Blood agar, MacConkey agar• Inoculation: Quadrant streak plate technique• Incubation: Normal atmosphere, 36-/+2ºC,overnight

Throat• Medium: Blood agar• Inoculation: Quadrant streak plate technique,by stabbing the agar.• Incubation: Normal atmosphere, 36-/+2ºC,overnight

Sputum (will be shown on day 2)• Medium: Blood agar, MacConkeyagar, chocolate agar• Preparate: two• Inoculation: Quadrant streak platetechnique• Incubation: candle jar

Incubation• Appropriate temperature– 35-37°C• Appropriate atmosphere– Aerobe (incubator)– %5 CO 2 (candle jar or incubator with %5 CO 2 )– Anaerobe– Microaerophilic• Appropriate time– 16-18 hours (one night)– 24 hours– 48 hours– 7 days– …

First day• Front bench– Containers• Sterile urinecontainer• Non-sterilecontainer• Sterile falcon tube• Transport medium• Each bench– Clinical samples• Urine: Blood and MacConkeyagar• Stool: MacConkey and SS agar• Pus: Blood and MacConkey agar• Throat: Blood agar– Media to be inoculated• Blood agar• MacConkey agar• SS agar

Inoculation of clinicalsamples and evaluation ofculturesSecond day

Evaluation of the cultures• Growth of the causative agent ofinfection• Identification of the microorganism• -/+ antibiotic susceptibility testing

Evaluation of direct smear

Evaluation of direct smear

Evaluation of direct smear

Evaluation of direct smear

Evaluation of Culture Plate• What is the sample? Is the sample sterile?Has it been taken from a body site whichhas normal flora?• Any growth?• Colony count? (for quantitative inoculation)• How many types of bacteria?• If we are seeking a certain pathogen, do anycolonies have characteristics of it?

Evaluation of Culture• Macroscopic examination of colonies• Microscopic examination of colonies• Manual tests• Automated or semi-automatedidentification systems• Antibiotic susceptibility testing

Macroscopic Examination ofColonies• Colony morphology• Differentialcharacteristics ofthe media:– Blood agar:Hemolysis– MacConkey agar:Lactose utilization– SS agar: H2Sproduction

Hemolysis• Blood agar– Alpha-hemolysis– Beta-hemolysis– Gamma-hemolysis

Lactose utilization• Mac Conkey agar– Lactose positive– Lactose negative

H2S production• SS agar:– H2S positive– H2S negative

Microscobic examination of the• Gram stainingcolonies

Manual tests• Catalase• Coagulase• Oxidase• CAMP• Growth at 6.5% salt medium• Growth at Bile-esculin medium• Optochin susceptibility• Bacitracin susceptibility• Biochemical tests:– TSI(triple sugar iron) medium– IMVIC– Urease test– …

Automated or Semiautomatedidentificationsystems

Antibotic Susceptibility Tests

Second Day• Front bench– Ready made slidesfrom sputum samples– Sputum culture plates• Each bench– Evaluation ofculture plates– Gram staining

Gram staining•Crystal violet (1 min)•Wash with water•Lugol (1 min)•Wash with water•Alcohol (30 sec)•Wash with water•Fuchsin (30 sec)•Wash with water