8/8/2019 Micro Ex 7-9

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Ex7:Action of Disinfectants on Microorganisms Ex8: Antimicrobial Susceptibi li ty Test: Diffusion Test ProcedurePurpose :

study the bactericidal effect of concentration and time of exposure todisinfectants on bacteria

determine the relative effectiveness of certain standard disinfecting agents

▪ To show in-vitro sensitivity of different bacteria to selected antibiotics

Organism used :Escherichia coli Different Bacteria used.Materials :

5 lactose broths in Durham’s fermentation tubes

Disinfectants UsesPhenol 5%Lysol 5%

distorts CM

BetadineMerthiolateSodium hypochlorite 1%H2O2 3%

protein oxidation

Mercuric chloride 1:1000 enzyme inactivationIsopropyl alcohol 95%Ethyl alcohol 70%

CHON Coagulation

- Zephiran chloride,1:1000

Zephiran chloride 1:1000 Damages CM &denatures CHON

Mueller Hinton Infusion Agar (MHIA)▪ Gives satisfactory growth of the majority of rapidly growing pathogens▪ Not antagonistic to the major antimic agents to be tested▪ Isotonic w/ blood&w/ the addition of 5% defibrinated blood

BaSO4 Comparison Standardcell density : 1.5x10 8 CFU/mL organisms

Procedure : [important steps]After an interval of 5’, 10’, 15’ and 30’, transfer from inoculated disinfectants

to lactose broth tubesIncubate at 37°C for 24hrs.Observe bacterial growth.

growth: pink color in test tube; due to Andrade’s indicator—turns pink inthe presence of acid

bacteria will ferment lactose acid and gas

gas production: presence of bubbles and displacement of medium in thesmall inverted tube

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▪ Inoculate until turbidity matches that of the BaSo4 Comparison Standard▪ Dip sterile swab into standardized suspension. Remove excess fluid bypressing and rotating the swab against side of tube above fluid level.

MoI: very close streaking—3 direction streaking over the entiresurface of agar, turning 60° each time

▪ Let stand for 3-5 minutes (not >15mins)▪ Apply antibiotic impregnated discs (sterile forceps)▪ Gently press down to insure complete contact

Special arrangement: no closer than 15mm to the edge of the plate and farenough to prevent overlapping zones of inhibition(atleast 20mm apart)

▪ Incubate at 37°C for 16-18hrs.

Ex 9: StaphylococcusPurpose :

To acquaint the student w/ diff char’s of Staphylococcus To differentiate bet pathogenic and nonpathogenic Staphylococci

Organisms used: S. epidermidis and S. aureus

Materials :Blood Agar plate |Trypticase Soy Agar |Mannitol Agar |Plasma |NSS |H 2O2

Procedure:Inoculate BA plate w/ 4Q streaking. TSA: simple streaking

Tests Performed Detects Action Result

Slide Coagulase Bound coagulase Bound coagulase binds plasmafibrinogen

Clumping w/in 10secs

Tube Coagulase Free coagulase Free coagulase activates plasmafibrinogen

Clotting intervals of 30mins. for 4hrs; if no clot examine- 6&24hrsCoagulum- weakly +Complete clotting- strongly +

Mannitol Fermentation med used: Mannitol agar Reaction : Acid – pink ; Alk- yellow ; Negative- colorless

Catalase Production H2O2→catalse→H2O+O2 + gas bubbles- resul t of the presence of O2 or gas

S. Aureus S. epidermidisCatalase + +Coagulas

e + -

Mannitol + -

Pigment Goldenyellow White