Microbiology Lab practical: Rapid Microbial Testing & Identification for Gram Positive organisms

What is source specificity?

Refers to what gives the test its ability to detect a specific pathogen or antigen and NOT interact with others

Source Specificity in Rapid Strep

This test detects Group A streptococcus pyogenes

• Specificity comes from antibodies on the test strip that bind to Group A carbohydrate antigen present on the bacterial cell well

Source Specificity in Rapid Staph

Identifies staphylococcus aureus

• Specificity comes from detection of Protein A and/ or clumping factor (coagulase)

• This interactions cause visible agglutination if          S. aureus is present (positive result)

Rapid Strep testing process

Tests for presence of Streptococcus pyogenes

• Reagents are drops on swab in a tube then a strip is placed in the solution

• The flow of the reagent and bacteria solution flow upwards 1st hitting the test line, then the control line

• Both lines have to be red for the rapid test to positive

• One line on either= negative

What’s going on here?

1. 1st strip is positive because BOTH the test and control line are red

2. 2nd strip is negative because their is only one red line at the control

3. 3rd strip is invalid because there is a missing control line

Rapid Staph testing process

Tests for presence of Staphylococcus aureus

• Identifies Protein A & coagulase

1. 1 drop of Test latex on BOTH circles (sample and (-) control circle’s)

2. 1 drop of (-) control on the control circle

3. Add culture to sample circle and mix to see results

4. Agglutination @ Sample circle= POSITIVE

5. If there is agglutination on the negative control circle, the test is INVALID

• Negative control circle should NEVER have agglutination!!!!

What does this image mean?

The rapid staph test is Positive because there is agglutination on the sample/ test circle

• Positive for Staphylococcus aureus

Catalase Test

This test helps identify if the organism will liberate Oxygen gas or not (bubbling or not)

• Converts hydrogen peroxide to O2 and H2O

• Positive= bubbling occurs→ continue with catalase (+) tests

• Negative= NO bubbling occurs→ continue with catalase (-) tests

What tests do the Catalase positive bacteria go through?

1. Novobiocin Sensitivity and Growth characteristics of MH agar

2. DNAse agar

3. MSA agar

4. PYR test

What tests do the Catalase negative bacteria go through?

1. Sensitivity to the antibiotic Bactrim (SXT) and hemolysis (Blood agar and disc)

2. Esculin Hydrolysis in the presence of bile (BEA Slant)

3. PYR(Broth) test

Novobiocin Sensitivity (Growth characteristics) of MH agar

1. Swab culture and put onto the MH plate (one side of plate filled with culture & other side do the streak method)

2. Put an antibiotic disc on the “streaked” slide 

• Bacteria growing closer to the disc= Resistant to Novobiocin; larger zone of inhibition

• Bacteria growing away from disc= Sensitive to Novobiocin

What is going on here?

Novobiocin Sensitivity: An antibiotic disc is place here to see if the bacteria is susceptible or resistant to the disc

• There’s a large zone of inhibition

• Growth away from disc

• Result= Resistant

DNAse agar

This plate differentiates b/w organisms ability to secrete exoenzyme DNAse

• Positive result= clear zone and does contain the exoenzyme DNAse

• Negative result= no clear zone and bacteria doesn’t contail DNAse

What does this mean?

There is no zone of clearance therefore this organism does NOT secrete the exoenzyme DNAse= NEGATIVE 

MSA agar

• Selective for halotolerant organisms (media contains 7.5% NaCl)

• Differential is organisms can ferment mannitol or not

• Yellow color change= organism can ferment mannitol (a type of carbohydrate substrate) LOWer pH

• No color change= bacteria is a non-mannitol fermenter (takes on color of media)

PYR test

Checks to see whether bacteria produce the enzyme pyrrolidonyl arylamidase (PYRase) by dropping the PYR reagent onto the swab with the culture

• Red/pink= contains the PRYase enzyme

• Yellow= Doesn’t contain the PYRase enzyme

SXT sensitivity and hemolysis

Only done if the catalase test was negative

• is bacteria sensitivity to the antibiotic Bactrium (SXT)?

• Blood agar is used here (enriched and differential among streptococcus species)

• Types of hemolysis

• S or R

BEA slant

Selective and differential medium

• Can organism hydrolyze carbohydrate esculin

• Positive for esculin hydrolysis= black color within slant (more than 50%)

• Negative=less than 50% of slant is back, or there’s no black

What does this slant mean?

This is a BEA slant

• Black color= able to hydrolyze esculin

What are the blanks?

Positive or Negative for Catalase test

• Positive for catalase test is the top

• Negative or Catalase test is the bottom