Unknown ID Staphylococci
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Staphexerciseset-PPt1.pptxBIOL 2420
Identification of Staphylococcus Species
Objectives:
To learn the characteristics of Staphylococcus
To learn how clinical specimens are collected
To learn the uses of selective and differential agar to determine characteristics of bacteria
To be able to identify different types of hemolysis
To understand how to use different tests to narrow down and presumptively identify staphylococcal species
How to collect a nasal Staphylococcus sample:
Watch the following videos:
Clinical setting showing proper handwashing, labeling, and collection
Example of a nasal swab procedure (the procedure takes place at 0:20s)
Picture credit: St. Luke’s Regional Laboratories
Test your sample on an MSA plate
Mannitol Salt Agar (MSA)
What makes this plate selective?
What makes this plate differential?
Name the indicator and how it works.
Why do we use this plate when testing Staphylococcus?
What was the purpose of doing a streak plate with your nasal sample?
What are some concerns with handling patient samples?
Plate from mkldiagnostics
Uninoculated
After incubation:
Explain the MSA plate result
Picture credit: mkldiagnostics
Uninoculated
24-48 hour incubation
Hemolysis and Novobiocin sensitivity tests
Columbia nalidixic agar (CNA) plates
What makes the plate selective?
Why is this important to help presumptively identify this bacteria as Staph?
What makes the plate differential?
Staph species are either ___- or ___- hemolytic.
Explain these two types of hemolysis.
Novobiocin
What is a zone of inhibition?
What does it mean if your bacteria has a zone of inhibition around the antibiotic tab?
How many mm must the zone of inhibition be for the Staph to be considered sensitive?
After incubation:
Using Plate #1:
Name the hemolysis.
Give the definition of this type of hemolysis.
Using Plate #2:
How do you measure a zone of inhibition?
What size is the zone of inhibition?
Is the presumptive Staph sensitive or resistant to novobiocin?
How many mm is considered sensitive?
Before incubation:
Streaked CNA plate with Novobiocin tab in Quadrant 1
After incubation Plate #1
After incubation
Plate #2
Now that you have an isolated presumptive Staphylococcus species:
Gram stain to confirm cell morphology
By what cell structure does does the Gram stain differentiate bacteria?
Can you list the reagents used in the Gram stain? Explain the purpose of each.
Give the Gram reaction.
Gram + or Gram -?
Describe the cell morphology.
Now that you have an isolated presumptive Staphylococcus species:
You will perform a catalase test
Describe the procedure you would do in the lab.
What enzyme does this test test for?
Do you use an indicator or reagent? Name it.
Is the result catalase + or catalase -?
Now that you have an isolated presumptive Staphylococcus species:
Does your sample meet all the criteria to be a presumptive Staphylococcus?
Check your answers against the following criteria.
Gram +
Cocci (spheres)
Clusters (staphylo-)
Catalase +
Halotolerant
Either beta- or gamma-hemolytic
If you answered yes to all the above information, continue to the next slides.
Is your presumptive Staphylococcus a potential pathogen?
SM110 agar
What makes this plate selective?
What makes this plate differential?
How do you differentiate a potential pathogenic Staphylococcus from a potentially non-pathogenic Staphylococcus?
You inoculated the plate on the right with your sample. Is the bacteria SM110+ or SM110-?
24-48 hour incubation
Picture adapted from mkldiagnostics catalog
Is your presumptive Staphylococcus a potential pathogen?
Deoxyribonuclease (DNAse) agar
What exoenzyme does this plate test for?
What is an exoenzyme?
Name the indicator.
Name the substrate present in the agar that will be broken down by the enzyme.
Give describe what a positive and negative reaction look like.
Is your sample DNAse + or DNAse -?
24-48 hour incubation
Methyl green DNAse agar used in the lab does not need HCl
Is your presumptive Staphylococcus a potential pathogen?
In lab, you would have been asked to determine if your sample is both SM110+ and DNAse + before doing the following test.
However, we will “perform” it here.
Coagulase test
What exoenzyme are we testing for?
If your bacteria produces this enzyme, is it more or less likely to be pathogenic?
How does the production of this enzyme increase pathogenicity?
Name the media.
Is there an indicator or reagent?
Is your sample coagulase + or coagulase -?
Uninoculated
24-48 hour incubation
Use your test results and the provided charts to determine the species of your Staphylococcus
What is the most likely species of your Staphylococcus?
NB
NB
N B
25 m
m
N
B
2
5
m
m
