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Bacteriology 1 Lab 1

Staphylococci Gram + Cocci Cluster in arrangement

Gram stain

mannitol salt agar MSA selective and differential growth medium high concentration (about 7.5%-10%) of salt contain carbohydrate mannitol Ph indicator : phenol red Staphylococcus aureus produces yellow colonies S.epidermidis S. aureus

Catalase test Procedure:- 1- add few drops of H2O2 on a slide Is used to differentiate between staphylocci & streptococcci Staphylococci give positive result Use 3% H2O2 (hydrogen peroxide )as substrate gas bubble is produced for positive results Procedure:- 1- add few drops of H2O2 on a slide 2- transfer a small amount of colony growth to the slide 3- observe the bubbling reaction

Coagulase test Is used to differentiae between S.aureus and other staphylocci species. S.aureus gives positive reaction Fibrinogen (plasma) is the substrate Fibrin (visible clot) is produced

DNase test Is used to differentiae between S.aureus and other staphylocci species. DNA is the substrate The hydrolysis of DNA is detected by observing a zone of clearance Procedure:- 1- Using a sterile loop, inoculate organisms on Dnase plate. Make sure each test area is labelled clearly. 2- Incubate the plate aerobically at 35oC for 13 to 24 hours. 3- Flood the surface of the plate with 1 N hydrochloric acid (HCL )solution. Tip off the excess acid. 4- Look for clearing around the colonies within 5 minutes of adding the acid. zone of clearance

Summary + - Tests S.aureus Other Staphylococci Catalase test Coagulase test - Dnase test

Lab 2+3 streptococci

Streptococci Gram + cocci Chains in arrangement Catalase negative

Types of hemolysis : Blood agar

Alpha hemolysis Partial hemolysis of RBCs Mainly S.pneumoniae & S. viridans To differentiate: optochin disc S.pneumoniae is sensitive while S. viridans is resistant.

Beta- hemolysis Complete Hemolysis of RBCs Mainly GAS (Group A streptococci ) & GBS (Group B streptococci ) To differentiate: bacitracin test GAS is sensitive while GBS is resistant.

Gamma – hemolysis No hemolysis Mainly Enterococci : E. faecium & E. feacalis Can grow at 6.5 % salt NACL Can hydrolyze esculin in 40% bile ( bile esculin) Bile Esculin agar - Bile is the selective agent - Esculin is used to differentiate Enterococci from other bacteria BILE inhibits gram + bacteria except enterococci & group D streptococci

AFB Lab 5

Gram stain Gives positive or negative reaction It does not applied to all bacteria ex. Acid Fast Bacilli

Acid Fast Bacilli (AFB) Why Gram stain is not used ? - AFB cell wall has ~ 60% lipid (waxy). So, it makes gram stains impermeable. Ex. Mycobacteria spp. Mycobacterium tuberculosis (MTB)

AFB stain Two procedures : 1- Light microscope - Ziehl-Neelsen and Kinyoun methods 2- Fluorescent microscope - Fluorochrome method

Ziehl-Neelsen stain Procedure :- 1- Primary stain: carbol fuchsin , 5 minutes( basic fuchsin +phenol) 2- decolorizer : acid alcohol , 1-2 minutes 3- counterstain: methylene blue, 1 minutes

Ziehl-Neelsen (ZN)staining Hot method by using heat Cold method ( no heat ) is called Kinyoun - increasing concentration of basic fuchsin and phenol

Summary Hot ZN staining Procedure :- 1- Primary stain: carbol fuchsin (basic fuchsin +phenol) , 5 minutes 2- decolorizer : acid alcohol , 1-2 minutes 3- counterstain: methylene blue, 1 minutes

Neisseria Lab 6

Neisseria Gram negative cocci - diplococci (bean shaped)

Biochemical reaction Catalase +ve Oxidase +ve

Oxidase test The Oxidase Test is used to identify bacteria containing the respiratory enzyme cytochrome c oxidase TMPD

culture Chocolate medium + 5% CO2 (capnophilic) Incubation: At 37ºC for 24-48 hrs. Colonial morphology: small, gray, translucent and raised.

Neisseria spp. : Carbohydrate Utilization test only one carbohydrate source in the medium Phenol red as pH indicator Differentiate between N.gonorrhea & N.menengitidies

Neisseria spp. : Carbohydrate Utilization test N. Gonorrhea N. Meningitides Glucose Maltose Lactose N. Gonorrhea + - N. Meningitides

Bacillus Lab 8

bacillus Gram positive Rod Spore forming

Bacillus subtilis Culture : - nutrient agar : creamy irregular colonies - Blood agar : Beta-hemolysis

Endospore staining

Bacterial Endospores Endospores are a dormant stage of some bacterium that allows it to survive conditions that would normally kill bacteria such as extreme drought or heat Endospores ultimately function are protection for the bacterial genome Endospores provide resistance against: Low nutrient conditions Radiation High temperatures and various chemical disinfectants

The Vegetative Cell Gives Rise to One Spore Bacterial Cell Spore Bacterial Cell The endospore is able to survive for long periods of time until environmental conditions again become favorable for growth. The endospore then germinates, producing a single vegetative bacterium.

Not all bacterial species can form spores A few genera of bacteria produce Endospore such as Clostridium and Bacillus (Endospore production is associated with Gram Positive bacteria)

Schaeffer-Fulton Stain Procedure 1. Prepare a smear. Air Dry. Heat fix 2. Put the slide on steam rack 3. Flood the smear with Malachite Green stain 4. Steam slide for 5 minutes (be sure that the satin is not dry, if so , add more satin ) 5. Drain slide and rinse with DI water. 7. Flood smear with Safranin (counter stain). This stains the vegetative cell. (Leave for 1 minute) 8. Drain the slide and rinse with DI water 9. Dry 10. View slide under 100x oil immersion.

Endospore Stain Example Spores: Green Cell: Red or Pink Practical: Each student will make smear of Bacillus subtilis