Midterm

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Enterococcus: Classification and Habitat

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Formerly group in Group D Streptococci. 12 species. Now its own genus. Found in the GI tract.

Used to indicate fecal contamination of water.

1) E. faecalis
2) E. faecium

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Enterococcus: Characteristics

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Resemble Streptococci in colony and cell morphology. Can be alpha, beta, gamma (most common type) hemolysis on Blood Agar Plates.

All bile esculin positive and grow in 6.5% NaCl broth.

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Enterococcus vs. Streptococcus

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Enterococci can hydrolyze esculin in the presence of 4% bile.

Esculetin reacts with iron salt to from a dark brown or black complex.

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Bile Esculin Medium

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Used for identification of enterococci and nonenterococci (Group D) has ferric citrate as the source of ferric iron. Medium turns dark when above-mentioned bacteria are streaked on it.

4% bile in the medium can inhibit some bacteria (streptococcus).

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Nonenterococci

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Bile esculin positive, but do not grow in 6.5% NaCl.
Enterococci are less susceptible to penicillin than nonenterococci.

1) Streptococcus bovis
2) Streptococcus equinus

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Pathogenic Bacteria of the Throat/Nose Include

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1) Streptococcus pyogenes (Group A Streptococcus)
2) Streptococcus pneumoniae
3) Staphylococcus aureus
4) Corynebacterium diptheriae
5) Neisseria gonorrhoeae
6) Neisseria meningitides

Candida (Yeast) can also be isolated.

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Nonpathogenic Bacteria of the Throat/Nose Include

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1) Streptococci viridans (alpha hemolytic)
2) Staphylococcus epidermidis
3) Neisseria
4) Corynebacterium (diphtheroids)

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Methods to Identify Nose/Throat Isolates

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1) Hemolysis
2) Coagulase Test
3) Bile Solubility
4) Oxidase Test
5) CTA Sugars
6) CAMP Test
7) Optochin/Bacitracin
8) Mannitol Salt Agar
9) Catalase Test

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Blood Agar Plates

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Used for hemolysis test. Needed as growth factor for some bacteria.

Made from TSA (tryptic soy agar) and blood agar base. Defibrinated sheep blood is preferred.

Sheep blood is preferred because Haemophilus hemolyticus (nonpathogenic bacterium similar to streptococci) is inhibited by this blood type.

Human blood contains inhibitory substances such as citrate and dextrose as well as some antibiotics, and H. hemolyticus is not inhibited by this blood type.

Horse blood can give incorrect hemolytic reactions.

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Blood added when Medium is Hot

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Known as chocolate agar. Can also be made by using 1% dehydrated hemoglobin instead of blood to the medium.

Neisseria gonorrhoeae.

Because blood cells are lysed, providing hemin (released from hemoglobin during lysis) and NAD (heat inactivates enzyme that hydrolyses this)

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Alpha Hemolysis

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Streptococcus Salivarius.

Produce colonies surrounded by 2 separate zones of RBCs. Green discoloration because heme iron is present in those cells become oxidized by H2O2.

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Beta Hemolysis

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Streptococcus Pyogenes.Wide, clear zone of hemolysis with no RBCs visible.

There are 2 types of beta hemolysins, and most bacteria demonstrate both types. Some only demonstrate type O, so stabbing into agar to streak is recommended.

1) Streptolysin O: Destroyed by oxygen and only found in subsurface colonies on blood agar plates.
2) Streptolysin S: Oxygen stable and responsible for surface hemolysis.

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Gamma Hemolysis

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Streptococcus bovis.

Produce colonies that are surrounded by no zones of hemolysis.

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Coagulase Test

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Used to distinguish Staphylococcus aureus from Staphylococcus epidermidis.

Coagulase is an extracellular enzyme made by S. aureus. Causes citrated plasma to coagulate and produce a clot. May be responsible for fibrin barrier and coating made by S. aureus.

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How to Perform Coagulase Test (2 methods)

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1) Tube Test: 0.5mL rabbit plasma and adding culture. Incubate at 37C and observe for 4 hours. Re-examine after 20 hours.

2) Slide Test: Bacterial growth added to suspension and mixed for 5 seconds. White clumps indicate positive reaction.

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Bile Solubility

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Streptococcus pneumoniae can be differentiated from other types of alpha hemolytic streptococci by its lysis of bile salts. Bile, SDS act on the cell wall of S. pneumoniae by activating L-alanine aminase which lyses the cell.

S. aureus used as negative control.

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How to Perform Bile Solubility Test

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Add 10% SDS to cells placed on slide from colonies on an agar plate. Observe cell dissolution.

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Oxidase Test

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Tests for cytochrome C oxidase (respiratory enzyme responsible for reducing water during the electron transport chain. Neisseria and pseudomonas are positive.

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How to Perform Oxidase Test

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1% tetramethyl-dihydrochloride on colonies. Oxidase positive turn pink, then maroon then dark red and then black. Bacteria are killed by oxidase reagent.

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CTA Sugars

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Species of Neisseria and Moraxella catarrhalis can be differentiated with this. Examined with Cystine Trypticase Agar (CTA) which contains cystine to reduce the atmosphere in the medium.

Only done for oxidase positive gram negative diplococci.

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How to Perform CTA Sugars Test

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Inoculate CTA tubes containing 1% dextrose, sucrose or maltose with the bacterium. Incubated for 72 hours and observed every 24 hours. Sugars that are used will turn the pH indicator (phenol red) from red to yellow.

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CAMP Test

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Christie, Atkins and Much-Peterson. Used to identify Streptococcus agalactiae (GBS). Most GBS produce CAMP factor. Acts synergistically with beta hemolysis of Staphylococcus aureus.

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How to Perform CAMP Test

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Streak the streptococci onto a sheep blood agar plate and then streak beta hemolysis producing staphylococcus aureus perpendicular to, but not touching, the streptococci. Incubated under CO2. Zone of hemolysis means positive.

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Optochin Sensitivity and Bacitracin Differentiation

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Identify S. pneumoniae and S. pyogenes (GAS) respectively. Done on blood agar plate and incubated under CO2.

Optochin Test: 14-16mm is sensitive.
Bacitracin: Any zone is positive. Bacitracin is -.04 units, not 10 units.

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Mannitol Salt Agar

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Strains of staphylococcus aureus use MSA, while Staphylococcus epidermidis does not.

Appearance of yellow halo around bacterial colonies means mannitol utilization.

Bacteria that don't use mannitol but can grow in 7.5% NaCl from the medium won't turn the medium from red to yellow.

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Catalase Test

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Tests for the enzyme catalase. Staphylococcus is differentiated from streptococcus since staphylococcus makes catalase.

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How to Perform Catalase Test

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Drop of 30% H202 dropped on bacterial colony on a glass slide. Immediate bubbling as H2O2 is converted to O2 and H20 is positive. Careful not to get RBCs from blood agar plate since there also have catalase.

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Corynebacterium Diphtheriae Identification

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Examine colony morphology on blood-tellurite plates (colonies are black). Also have granules. Use glucose, not sucrose. Done on Loeffler Coagulated Serum. Pleopmorphic.

Nonpathogenic species of corynebacterium (pseudodiphtheriticum) are called diphtheroids and have no granules.

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Hydrolysis

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Not all bacteria have hydrolytic enzymes (turn big products into smaller products).

Starch is hydrolyzed by amylase (with Gram's Iodine, clear areas means positive, Non-hydrolyzed is blue).
Gelatin by gelatinase (liquification is positive).
Tween-80 by lipases. Yellow to pink (oleic acid production) is positive.
Casein by proteases (clear zones is positive).

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Bacteria that can Hydrolyze
[ ] Means reference culture.

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1) Bacillus cereus, [B. subtilis] and B. megaterium (starch hydrolysis).

2) P. aeruginosa, [B. subtilis] and S. aureus (gelatin hydrolysis)

3) [Mycobacterium smegmatis] and M. kansaii (Tween-80).

4) Nocaria brasiliensis, B. cereus, [B. subtilis] and B. megaterium (casein hydrolysis).

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Nitrate Reduction

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Reduce nitrate to nitrite. Almost all Enterobacteriaceae (E. coli used) reduce nitrate. Used to differentiate Haemophilus ducreyi and Haemophilus vaginalis (both don't reduce nitrates) from other Haemophilus (who do reduce nitrates). Moraxella catarrhalis and Neisseria mucosa (both reduce nitrate) can also be differentiated from other Neisseria (who don't reduce nitrates).

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Specifics of Nitrate Reduction

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Nitrate (NO3) to Nitrite (NO2) and to Nitrogen gas (N2) takes place under anaerobic conditions. Ammonia, nitric oxide and nitrous oxide can also be end-products.

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Denitrification

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Reduction of nitrate to N2 or nitrous oxide. Nitrate is electron acceptor. For each molecule of nitrate reduced, 5 electrons are accepted.

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How to Perform Nitrate Reduction Test

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Inoculate pure bacterial culture into a tube a nitrate broth containing potassium nitrate. Looks for disappearance of nitrate or appearance of gas (both positive).

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Colors of Nitrate Reduction

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5 drops of Nitrate Solution A and B and red in 1-2 means nitrate to nitrite.

If no red color, nitrate could have been reduced to nitrous oxide, ammonia or N2.

Zinc dust reduction test can be used to determine if nitrate still present in the broth. Red color mean nitrate present.

Inverted Durham tubes used to detect gas.

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Stains

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Made from either acidic, basic or neutral dyes.

Methylene blue is made of the salt methylene blue chloride.

Eosin stain made of sodium eosinate.

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Stains and Reference Bacterium

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1) Dorner and Malachite Green: B. subtilis
2) Acid-Fast: M. smegmatis
3) Nigrosin Stain: None
4) Congo Red: None
5) Capsule Stain: K. pneumoniae

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Spore Stain

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Dorner (spores red, colorless sporangia and black background) and Malachite Green (spores green in red sporangia). Endospore stain. Spprangia (vegetative cells) of Bacillus and Clostridium take up stain while spores remain unstained. Heat used to drive primary stain into the spores.

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Acid-Fast Stain

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Mycobacterium, Norcardia, Bacillus and Clostridium resist acid-alcohol decolorization. Ascospores of certains yeasts are also acid-fast. Retention of stain due to mycolic acids in cell wall.

Acid-Fast are pink/red, non-acid fast are blue.

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Negative (Relief) Stain

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Nigrosin and Congo Red. Background, not the organism is stained. used to show that cells don't take up stains easily. Cell morphology is not distorted.

Air dry.

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Capsule Stain

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Staining cell and background via a negative stain. Uses milk broth. Cells and surrounding background are purple. Capsules are unstained/blue and appear as a halo around the bacterium. Capsules don't really retain stain.

Do not heat fix.

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Polar Flagellated

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Flagella attached at 1 or both ends of the cell.

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Lophotrichous Bacteria

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Tuft of flagella at the end of the cell.

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Peritrichously Flagellated

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Flagella growing from many different places.

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Semi-Solid Agar

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Low agar concentration to allow bacteria to visually move. Contains TTC which is reduced when bacteria metabolize (becomes red when reduced). Red everywhere is motile, negative is red only along inoculation lines.

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Simple Mount and Hanging Drop

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Bacterial motility in real time. Not heat fixed. No stain.

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Flagella Stain

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Use young cultures since flagella is easily ripped off. Leifson method is used. Mordant, tannic acid used to thicken up the flagella to make it easier to view.

Reference is E. coli (peritrichous flagella), other uses P. aeruginosa (polar flagella).