Microbiology Lab: Exam 2

Covers labs 4-6

What organisms produce catalase? What type of bacteria is the Catalase Test specifically useful in identifying?

Aerobic microbes, specifically Staphylococci

What organisms do not produce catalase? What type of bacteria specifically does NOT produce catalase?

Obligate anaerobes and aerotolerant bacteria do not produce catalase, Streptococci specifically

What catalase is and what reaction does it perform? Why is this important?

it is an enzyme that converts hydrogen peroxide to water and oxygen gas. Hydrogen peroxide is a byproduct of metabolism in an aerobic environment and it is a highly reactive oxidizing agent that can damage proteins, RNA, and DNA in the cell if left alone

Give a basic outline on how to perform the Catalse test? What is a positive result? What is a negative result?

The test is performed by taking a small amount of bacteria and putting it on a clear plate, then adding 1-2 drops of hydrogen peroxide to each bacteria sample. Bubbles are a positive result and no reaction is negative

What is the importance of Staphylococcus aureus in human health and what diseases does it cause?

Staph aureus is the most pathogenic of the staphylococci group and it can infect the skin and many other tissues in the body. It can produce a wide range of harmful enzymes and toxins. Some of the toxins and enzymes include a toxin that kills WBCs, triggers fibrin clot formation, or can cause shock. There are also several strains of antibiotic resistant Staph like MRSA and VRSA. S. aureus can also release an enterotoxin as it grows in food that will make you sick by eating the food, even without having to grow in your GI tract. It can cause skin infections, sepsis, endocarditis, pneumonia, and bronchitis.

Define MRSA and why is this important in human medicine?

Methicillin resistant staphylococcus aureus. This is important in human medicine because they are very difficult to treat and can lead to sepsis

What does selective media mean?

Selective media means it has a chemical in it that inhibits the growth of certain microorganisms while allowing the growth of others.

What does differential media mean?

Differential media is media that does not inhibit the growth of any microbe, but has a chemical in it that makes microorganisms with particular traits appear different from each other.

What makes Mannitol Salt Agar differential?

 it has the sugar mannitol in it as well as the pH indicator phenol red. Bacteria that can ferment this sugar will make the agar around it acidic and turn it YELLOW as the pH drops

What is a halophile?

A bacteria that can tolerate salt which is important in the Mannitol Salt Agar

What type of media is Mannitol Salt Agar and what is it made up of that makes it that way?

It is selective and differential; It is selective because it has a high concentration of Salt that prevents the growth of all non-halophillic (salt tolerant) bacteria, It is differential because it contains Mannitol (a sugar) and Phenol Red (pH indicator that turns from red to yellow in acidic conditions)

Which bacteria grow on Mannitol Salt agar ?

All halophilic bacteria

What color would appear if Staph. aureus was grown on Mannitol Salt Agar and why?

YELLOW because pathogenic staph strains ferment mannitol and release acidic byproducts as a part of that metabolic process.

What color would appear if Staph. epidermidis appear on the Mannitol Salt Agar and why?

It appears PINK because it does NOT ferment mannitol and so the Phenol red remains pink

What is the Mannitol Salt Agar plate best for identifying?

It is best for identifying Staphylococcus aureus as this bacteria ferments mannitol

What bacteria does the Staph latex agglutination test detect?

Staphylococcus aureus

What are the steps of the Staph Latex Agglutination test? What is a positive versus negative result?

take a card with two circles; Add 1 drop of Test Latex Reagent to one circle and 1 drop of control reagent to the other; Mix in 1-2 colonies of chosen bacteria into the dot of latex reagent in the test circle and do nothing to the control circle; Gently swirl the drops of liquid around for 30 seconds. A positive result turns the circle blue with red clumps. A negative results has no change so the liquid remains purple and clear. 

What is the importance of the ‘control’ reagent in the Staph Latex Agglutination test and what would indicate an invalid test result?

Any agglutination in the control reagent would mean the test is invalid; It is important to have a control to make sure the reagent isn’t auto clumping to nothing

Which cellular parts are recognized by the reagents in the Staph latex test? (Aka how does this test actually work?) 

The latex particles in the reagent are coated with antibodies to specific antigens on the cell wall of S. aureus and these same latex particles are also bound to coagulase and Protein A. Agglutination and a color change will be visible when the antigen-antibody complexes are formed between staph aureus and the latex particles

What is an enriched media?

Media that has added nutrients in it to promote better/ faster growth of certain bacteria

Why is blood agar considered a differential media?

It is differential because it contains blood cells and if bacteria can perform hemolysis they will turn the agar clear or green

What type of media is blood agar? Why?

It is enriched and differential; It is enriched because the blood technically has extra iron and nutrients in it that allow for better growth of Strep and other pathogenic bacteria

What is hemolysin and which 2 types of bacteria produce it?

Hemolysin is produced by Streptococcus and Staphylococcus; Hemolysin is a toxin that can break down red blood cells

What are the different types of hemolysis reactions? What does each mean?

Alpha-hemolysins just break down hemoglobin which results in a greenish color of the agar; Beta-hemolysins fully lyse the red blood cell and break down hemoglobin leaving the blood agar fully clear; Gamma hemolysis just means no hemolysis so the organism doesn’t have hemolysin

What disease does Streptococcus pneumoniae cause and what type of hemolytic reaction does it perform?

Causes pneumococcal pneumonia and otis media and performs an Alpha-hemolysis reaction

What disease does Streptococcus pyogenes cause and what type of hemolytic reaction does it perform?

Causes bacterial pharyngitis aka strep throat and performs a Beta- hemolysis reaction

What disease does Streptococcus (Enterococcus) faecalis cause and what type of hemolytic reaction does it perform?

It is one of the leading causes of hospital acquired infections and performs a Gamma hemolysis reaction/ does not do hemolysis

What is the gold standard for diagnosing strep. throat and why?

Blood agar plates are the gold standard for diagnosing bacterial pharyngitis because they have a lower rate of false negatives

What bacterial component is detected in the Strep Latex Agglutination test?

It detects carbohydrate antigens on the cell walls of streptococci and specifically looks for the Beta-hemolytic streptococci of the Lancefield Groups A and B

What is the procedure of the Strep Latex Agglutination test?

• Get 1 empty  tube

• Add 2 drops of both Reagent 1 and 2 into the test tube 

• Add 5-10 colonies of 1 bacteria that you want to test into this tube

• Add 4 drops of Reagent 3 into the same tube

• Obtain a test card and make 2 circles; 1 labeled A and the other B

• Add 1 drop of Latex A into the circle labeled “A” and add 1 drop of Latex B into the circle labeled “B”

• Mix in the bacteria in the test tube into each circle, swirl them around only for 60 seconds

• Record if any light blue clumps form

What is a positive, negative and invalid result for the Strep Latex Agglutination test?

Positive (+) - Latex A and Latex B show no agglutination; indicates it is Beta hemolytic Streptococcus

Negative (-) - Latex A shows agglutination, Latex B shows no agglutination (Streptococcus pyogenes) OR Latex A shows no agglutination, Latex B shows agglutination (Streptococcus agalactiae)

Invalid (o) - BOTH Latex A and Latex B show agglutination

Define Nosocomial infections

 Hospital acquired infections

Name the 3 most common categories of nosocomial infections

Urinary tract infections, blood infections, and pneumonias

What is ventilator associated pneumonia?

A type of pneumonia typically seen in patients on mechanical ventilators that is most often caused by Streptococcus and Staphylococcus

Why is MacConkey agar considered a selective media?

It inhibits the growth of gram-positive bacteria while allowing for the growth of gram negative bacteria because it contains bile salts and crystal violet

Why is MacConkey agar considered a differential media?

because it contains lactose and the pH indicator neutral red

What is the pH indicator in MacConkey agar and what color does it turn if an acid is present?

The pH indicator is neutral red and it turns from pink to dark red in acidic conditions

What type of bacteria is MacConkey Agar most useful to identify?

It is most useful in identifying enteric bacteria

What is the Oxidation- Fermentation test used for and when is it most useful?

This test is used to determine a bacterial species ability to oxidatively metabolize a carbohydrate as opposed to fermenting the same carbohydrate. This info is useful in the classification of Gram-negative bacteria.

What are the 2 pathways that bacterium metabolize carbohydrates?

Oxidative metabolism or fermentative metabolism

What does oxidative metabolism produce as an end product? What does fermentative metabolism produce as an end product? Which one is more acidic

oxidative produces CO2 while fermentative produces an acid which is more acidic than CO2

What pH differences do the colors of bromthymol blue indicate (Oxidation-Fermentation Test) ?

Yellow means acidic, green means neutral, blue means basic

(Oxidase Test) What is cytochrome c oxidase?

Cytochrome oxidase is an enzyme that reduces oxygen to water in the electron transport, it is the final enzyme

What does the Oxidase test detect?

The test detects the presence of Cytochrome c oxidase and can be used to distinguish gram- negative rods. 

What is the Oxidase test used for identifying?

Pseudomonas aeruginosa which contains Cytochrome c oxidase. Other Enterics will test negative.

What is the procedure for the Oxidase test? How do you interpret the results (pos/neg)?

• Draw two circles on a piece of paper towel

• Add oxidase reagent to each circle

• Add each organisms to a circle using a plastic loop

• Wait 1 minute for a reaction to occur, the area smeared with cells that are oxidase positive will start to turn blueish purple and progressively get darker and the negative sample will have no change. 

• NOTE: if a blue ring forms around the sample, not in it, it is a false positive and it is simply the reagent reacting with the paper

What are the similarities between MacConkey agar and Mannitol Salt agar? What are the differences? What is different about their general purposes?

They are both selective Mannitol salt agar has a bunch of salt in it so only halophilic bacteria can grow on it and MacConkey has crystal violet and bile salts which select for gram negative bacteria. They are both differential as well, but MacConkey differentiates based on gram-negative bacteria's ability to ferment lactose while Mannitol Salt Agar differentiates based on a bacteria’s ability to ferment Mannitol.  MacConkey is generally for identifying enteric bacteria while Mannitol is mainly for distinguishing Staphylococci, specifically Staphylococcus aureus.