Bacteriology - Biochemical identification and Antimicrobial Susceptibility testing

Haist

1. specimen checking

2. gram staining

3. inoculate

4. identification of genus and species

5. Susceptibility testing

What is the flow of laboratory procedure for the diagnosis of infectious diseases?

To obtain isolates

What is the purpose of inoculating bacteria?

• Blood agar

• MacConkey

• Chocolate

• Broth (thioglycolate)

Common plates or mediums that are used for inoculating?

• Staphylococcus spp.

• Streptococcus spp.

For cocci bacteria, yielding a Gram (+) gram stain indicates what?

• Neisseria spp.

For cocci bacteria, yielding a Gram (-) gram stain indicates what?

Catalase

the Staphylococcus spp. and Streptococcus spp. bacteria will be subjected to what test after gram staining?

• (+) = Staphylococcus Spp.

• (-) = Streptococcus spp.

Test result (Catalase)

• (+) = ?

• (-) = ?

30% hydrogen peroxide

Reagent used for the catalase test?

false - (+)

True or false: a (-) result in catalase test will produce effervescence/evolution of bubbles?

• catalase (convert hydrogen peroxide to water)

• Ability of the bacteria to produce catalase

Principle behind catalase testing?

can cause false (+); (iron can be oxidized)

Why is wooden stick preferred for Catalase test?

False - staphylococcus spp.

True or false: a streptococcus spp. will produce effervescence in catalase test

True - RBC can breakdown hydrogen peroxide

True or false: RBC (blood) may result to false (+) because it can breakdown hydrogen peroxide

• Water (H2O)

• Oxygen (O2)

2H2O2 subjected to catalase will yield to what?

coagulase test

After catalase test (+), what test is done after?

(+) = Staphylococcus aureus

(-) = Staphylococcus epidermis/saprophyticus

Coagulates test result??

• (+) = ?

• (-) = ?

Rabbit plasma with EDTA

Reagent for coagulase test?

Clot formation

Coagulation test is (+) is indicated by the formation of what?

the bound coagulase of S. aureus reacts directly with fibrinogen → precipitation of fibrinogen on the staphylococcal cell → clumping when bacterial suspension is mixed with plasma

Why does S. aureus yield a positive result in coagulase test (principle)?

Bound and free coagulase

How many coagulase does S. aureus produce and what are they?

Bound coagulase

clumping factor of S. aureus in coagulase test

Novobiocin susceptibility test

If coagulase test result is negative, what test is done?

testng if the bacteria is inhibited by anti-microbial

What is the purpose of susceptibility testing?

5 ug novobiocin

Reagent for novobiocin ST

Sensitve/Susceptible - S. aureus/S. epidermidis

Resistant - S. saprophyticus

Novobiocin ST result?

Sensitve/Susceptible - ?

Resistant -?

>16 mm

To be considered sensitive, zone of inhibition for novobiocin ST should be what?

MHA plate agar

What agar plate is used for novobiocin ST?

6mm; 5 ug of novobiocin

how big is the filter paper and how much novobiocin does it contain?

S. saprophyticus

Bacteria that is can grow or resistant to novobiocin?

16 hrs

How long should novobiocin be incubated?

Streptococcus sp

if catalase is negative, what bacteria can be seen?

• Optochin ST

• Bacitracin ST

• Growth in 6.5 NaCl

Test that are done if catalase test is negative?

Alpha-hemolytic

Optochin ST is used for what type of Streptococcus spp.?

Beta-hemolytic

Bacitracin ST is used for what type of Streptococcus spp.?

Gamma-hemolytic Group D

Growth in 6.5% NaCL test is used for what of Streptococcus spp.?

Alpha-Hemolytic.

type of hemolysis that indicates partial (green) hemolysis

Beta-Hemolytic.

type of hemolysis that indicates complete (clear) hemolysis

Gamma-Hemolytic Group D.

type of hemolysis that indicates no hemolysis at all

Taxo A

Type of bacitracin used for bacitracin ST?

Cell wall synthesis

What does Bacitracin inhibits?

0.04 ug (6mm)

How much bacitracin is in the filter paper?

Group A

(+) susceptible = Streptococcus pyogenes

(-) resistant = Streptococcus agalactiae

Bacitracin ST:

Group ?

(+) susceptible = ?

(-) resistant =?

(+) = > 10 mm (susceptible)

(-) = <10 mm (resistant)

ZOI measurement (Bacitracin)

(+) = ?

(-) = ?

b-hemolytic Streptococcus

Bacitracin ST is used for what hemolytic group of bacteria?

b-naphthylamine

What is produce if the enzyme l-pyrrolidonyl arylamidase reacts (hydrolyzed) with l-pyrrolidonyl-b-naphthylamide?

n,n-dimethylaminocinnamaldehyde; bright red precipitate

What is used to detect b-naphthylamine and what color is produced?

n,n-dimethylaminocinnamaldehyde.

What is the reagent used for PYR test?

(+) = Red color

(-) = yellow color or no color

PYR test? Color

(+) = ?

(-) = ?

(+) = Streptococcus pyogenes, Enterococcus faecalis

(-) = Other Streptococci

PYR test (what bacteria is present?)

(+) = ?

(-) = ?

• sodium hippurate (substrate)

Reagent used for Hippurate hydrolysis test?

Ninhydrin

What is the indicator for Hippurate hydrolysis?

sodium benzoate and glycine

What is produce if Hippuricase hydrolyzes hippurate

false - beta-hemolytic streptococci

True or false: hippurate hydrolysis test is used for a-hemolytic streptococci

deep purple color

What color is produce if ninhydrine is added?

Streptococcus agalactiae to other beta-hemolytic streptococci

hippurate hydrolysis is used to differentiate what bacteria?

(+) = Streptococcus agalactiae

(-) = Streptococcus pyogenes

Hippurate Hydrolysis test (bacteria it differentiates)

(+) = ?

(-) = ?

(+) = Deep purple color

(-) = Colorless/yellow/pink

Hippurate hydrolysis (color produced)

(+) = ?

(-) = ?

Christie, Atkins, and Munch-Peterson (CAMP)

What does CAMP stands for?

Arrowhead

What shape is produced if CAMP is positive

S. agalactiae; lysis of sheep RBC by staphylococcal B-lysin

CAMP Principle: ? produces CAMP factor that enhances the ? of ? by ?

S. aureus

What is used as control for CAMP?

(+) = Streptococcus agalactiae

(-) = Staphylococcus pyogenes

CAMP result (bacteria confirmation)

(+) = ?

(-) = ?

b-lysin

CAMP identifies bacteria that produces what?

Blood agar plate (sheep’s blood)

What agar plate is used in CAMP ?

(+) = Enhanced hemolysis (arrowhead - zone of beta)

(-) = no enhancement of hemolysis

CAMP Result (shape/pattern)

(+)=?

(-) = ?

Alpha-hemolytic group

What hemolytic group is identified in Optochin test

Atpase and production of ATP

What does the optochin interferes the production of what?

Taxo P

Optochin is also called (type of optochin) ?

ethylhydrocuprein hydrochloride

Optochin filter paper is composed of ?

(+) = Streptococcus pneumoniae

(-) = Staphylococcus pyogenes

Optochin STT (Bacteria)

(+) = ?

(-) = ?

(+) = >14 mm (6mm) disc or >16 mm (10mm disc) (Susceptible)

(-) = no zone of inhibition (resistant)

Optochin STT (ZOI)

(+) = ?

(-) = ?

True’

True or false: Gram (+) Group D Streptococci are inhibited by bile salts

bile (4%), esculin to esculetin

Organism capable of growth in the presence of ? and able to hydrolyze ? to ? will demonstrate growth

(+) = Group D Streptococci

(-) = S. pyogenes

Bile esculin test (bacteria)

(+) = ?

(-) = ?

(+) = growth; blackening

(=) growth and no blackening of medium; no growth

Bile esculin test (growth pattern)

(+) = ?

(-) = ?

lyses pneumococcal colonies

Bile or solution of bile salt does what to colonies?

lower surface tension, medium, accelerating, autolytic process

Bile salts ? between the bacterial cell membrane and the ?, thus ? the organism’s natural ?

Sodium deoxycholate

Reagent used for bile solubility test (tube)?

(+) = Streptococcus pneumoniae

(-) = Entereococcus faecalis

Bile solubility test ( test tube; bacteria)

(+) = ?

(-) = ?

(+) = clear solution or dissolved colonies

Testing positive in Bile solubility will result to what?

• Enterococcus

• Aerococcus

• Some species of Pediococcus

• Gram-positive cocci

Species that can withstand higher salt concentration?

Halophilic - salt loving

Gram positive bacteria is a what type of bacteria?

6.5% NaCl Nutrient broth

Reagent for salt tolerance test

(+) = Enterococcus spp.

(-) = other streptococci

Salt tolerance test (bacteria)

(+) = ?

(-) = ?

(+) turbid - presence of growth

positive salt tolerance test will indicate what?

Carbohydrate

• glucose - 0.1%

• lactose - 1%

• Sucrose - 1%

Peptone - 2%

indicator (acid production)

Fermenter

• acid/alkaline slant or acid butt and slant

Nonfermenter

• alkaline slant

• alkaline butt

• Indicator (H2S production)

Composition of triple sugar iron agar (TSI)

Phenol red

What is the indicator used for acid production?

ferrous sulfate

What is the indicator used in H2S production

aerobic/aerobes

the slant part of the butt-slant media supports what type of bacteria?

anaerobic/anaerobes

the butt part of the butt-slant media supports what type of bacteria?

For fermentation

What is the purpose of the carbohydrates in TSI?

False - gas production and acidic

True or false: if the bacteria can ferment, there is gas production and alkaline

Phenol red

What is the indicator for acid production?

True

True or false: if there is a presence of acid, phenol red will turn yellow

Ferrous sulfate

What is the indicator for H2S production

black

Ferrous sulfate will turn what color if positive?

(A) = yellow acid

(K) = ?red no acid (neutral)

Interpretation of TSI?

Phenol red

• (A) = ?

• (K) = ?

Peptone - neutralize acid

What is the purpose of peptone in TSI?

A- A/A

B - A/A

C - K/A

D - K/A

E- K/K

Interpretation of the tubes ?

Non-fermenter

What does K/K mean?