mcb lab prectical 2

What is a culture?

Multiplying microbial organisms by letting them reproduce in a predetermined culture media under controlled laboratory conditions.

What is a pure culture?

Single type of organism

What distinguishes agar from broth?

Agar is solid while broth is liquid

Selective media

Promote growth of some bacteria and/or limits growth of other bacteria.

Differential media

Distinguish between different bacteria based on changes in colonies or changes in media.

All-purpose/Supportive media

Contains nutrients that will support the growth of a large variety microorganism.

Enrichment media

Designed to promote the growth of a specific microbe present in low amounts within a mixed culture

What type of agar is TSA?

All-purpose/Supportive

What types of agar is EMB?

Selective and Differential

What is EMB selective for?

Gram - organisms

How does EMB differentiate between microorganisms?

Amount of lactose fermentation

What color is Escherichia coli on an EMB plate?

Metallic green

3 ways to obtain a pure culture

Spread plate, Pour plate, and Streak plate

Spread plate

Original culture is serially diluted then the final dilution is spread on the surface of a plate with a spreading rod. Surface colonies grow.

Pour plate

Original culture is serially diluted then the final dilution is added to molten agar. The molten agar/dilution is poured into a empty petri plate. Surface and subsurface colonies grow.

Streak plate

Original culture is directly diluted across an agar surface using an inoculating loop.

What are the differences between the pour plate method and the spread

plate method?

Pour plate is added to molten agar.

Where do the colonies grow in the pour plate versus spread plate method?

Surface and subsurface for pour. Only surface for spread.

What is a serial dilution?

Thin out a heavy population of bacteria across an agar surface.

What is the definition of a colony?

Group of bacteria, fungi, and other microorganisms grown on a solid agar medium.

How to label an agar plate?

Top edge of plate

What is the purpose of a streak plate?

To dilute or thin out the original sample until the organism of interest is isolated and pure.

Tryptic Soy Agar (TSA) Media

Contains nutrients that will support the growth of a large variety microorganism.

Mannitol Salt Agar (MSA) Media

Selective for halophiles/halotolerant and Differential to sugar mannitol

Eosin Methylene Blue Agar (EMB) Media

Selective for Gram negative organisms and Differential to lactose fermentation

What components in MSA make it both a selective and differential medium?

Mannitol and Salt

What is the pH indicator in MSA?

Phenol red

What components in EMB make it both a selective and differential medium?

Eosin Y and Methylene Blue

What does pink coloration on MSA plates indicate?

pH greater than 8.4

What does red coloration on MSA plates indicate?

pH between 6.9 to 8.4

What does yellow coloration on MSA plates indicate?

pH lower than 6.9

What does metallic green coloration on EMB plates indicate?

Large amount of lactose fermentation

What does dark blue or purple coloration on EMB plates indicate?

Small amount of lactose fermentation

Why is it important to do biochemical testing?

It can reveal information necessary to help identify bacteria within a sample

What is the carbon source in Simmons Citrate agar?

Citrate

What is the pH indicator in Simmons Citrate agar?

Bromothymol Blue

How does the pH indicator in Simmons Citrate agar work?

When the environment becomes more alkaline, the medium will change from a green to blue color.

Uninoculated Simmons Citrate slant

pH between 6.9 to 7.6, green agar slant, negative reaction

Inoculated Simmons Citrate slant

pH greater than 7.6, blue agar slant, positive reaction

What organism yield a positive reaction Simmons Citrate?

Enterobacter aerogenes

What organism yield a negative reaction Simmons Citrate?

E.coli

What type of medium is Urea and Simmons Citrate agar?

Diferential

Why is Urea a significant biochemical test?

The presence of the enzyme urease is a useful diagnostic test for identifying bacteria.

What is the pH indicator in Urea Agar?

Phenol red

How does the pH indicator work in Urea Agar?

When the environment becomes more alkaline the medium will change from a yellow color to fuchsia or hot pink.

Uninoculated Urea agar slant

pH 6.8 to 8.0, yellow agar slant, negative reaction

Inoculated Urea agar slant

pH greater than 8.0, fuchsia agar slant, positive reaction

What organism yield a positive reaction Urea agar?

Proteus vulagris

What organism yield a negative reaction Urea agar?

E.Coli

What is the chemical reaction for the decomposition of hydrogen peroxide?

2H₂​O_2​⇌2H_2​O + O_2​

What indicates a positive catalase result?

Rapid formation of bubbles

Between Staphylococcus and Streptococcus, which genus is positive for catalase activity?

Staphylococcus

Staphylococcus aureus Coagulase test result

Positive

Staphylococcus epidermidis Coagulase test result

Negative

Staphylococcus aureus Staphyloside test result

Positive

Staphylococcus epidermidis Staphyloside test result

Negative

Method used in lab to distinguish between S. aureus and S.epidermidis?

Coagulase test

What type of test is staphyloside test?

Latex

What antigen is in staphyloside test?

Protein A

Which panel shows agglutination in staphyloside test?

One with clumps or specks

What type of test is coagulase test?

Biochemical

What is coagulase and what does it do?

An exoenzyme that causes the fibrin of blood plasma to clot.

Where do Streptococcus species normally live on/in the body?

In oral cavity or urogenital cavity

Which Streptococcus species are of greatest medical importance?

Streptococcus pyogenes, Streptococcus pneumonia, and Streptococcus agalactiae

What disease does Streptococcus pyogenes cause?

Strep throat

What disease does Streptococcus pneumonia cause?

Pneumonia

Why is Streptococcus agalactiae an issue for pregnant women?

Causes neonatal meningitis and septicemia

What is Lancefield classification?

Separates streptococcus species based on unique proteins on the cell surface.

Group A Streptococcus

Beta hemolytic/complete clearing; Streptococcus pyogenes

Group B Streptococcus

Beta hemolytic/complete clearing; Streptococcus agalactiae

Group D Streptococcus

May be alpha, beta, or gamma hemolytic; Enterococci faecalis

Beta hemolysis

Complete hemolysis/destruction of red blood cells. Results in a clear area around the bacterial colony. Ex Streptococcus pyogenes

Alpha hemolysis

Partial hemolysis/destruction of red blood cells. Results in a greenish/brownish zone around the bacterial colony. Ex Streptococcus pneumoniae

Gamma hemolysis

Absence hemolysis/destruction of red blood cells. No reaction around the bacterial colony. Ex Enterococcus faecalis

What type of medium is Blood Agar?

Enrichment and Differential

Staphylococcus gram stain reaction

Posive

Staphylococcus shape/morphology

Cocci cluster

Streptococcus gram stain reaction

Positive

Streptococcus shape/morphology

Cocci chains

What rapid test can be used to differentiate between Staphylococci and Streptococcus?

Catalase test

What does the term “superbug” mean?

Bacteria resistant to most drugs

How does antibiotic resistance happen?

Bacteria evolve to resist antibiotics

What is MRSA?

Staphylococcus Aureus that is resistant to Methicillin and related beta-lactam antibiotics.

What is VRE?

Enterococci resistant to the antibiotic vancomycin and causes serious infections

What is ESBL?

Enterobacteriaceae that is resistant to Beta-Lactamase

What is DRSP?

Streptococcus Pneumoniae that is resistant to many drugs and causes bacterial pneumonia and meningitis

What is CRE?

Enterobacteriaceae that is resistant to Carbapenem

Which antibiotic resistant bacterium that is more severe than the rest?

MRSA

Epidemiology

Study of the spread of disease

Epidemiologist

Someone who studies the spread of disease

Index case

Patient 0, the initial patient to be infected in an epidemic

Who are the two main “think tanks” of epidemiology?

Center for Disease Control (CDC) and World Health Organization (WHO)

Air-borne transmission

By breathing in

Contact transmission

By touch or direct contact

Bloodborne transmission

By blood

Droplet transmission

Bu respiratory droplets such as saliva and mucus

Which transmission mode is more responsible for spreading disease?

Contact

T-Streak Procedure

1. Light your Bunsen burner and sterilize your inoculating loop.

2. Remove the cap and flame the opening of the test tube.

3. Dip the inoculating loop into the bacterial culture.

4. Flame the opening of the test tube and put the cap back on.

5. Hold the lid of the plate, pull your loop gently across the first section, back and forth along the top of the T

6. Put the lid back on the plate and sterilize the loop.

7. Rotate your plate 90º

8. Streak once through the first section you streaked and pull into the second section. Continue to streak the rest of the second section.

9. Put the lid back on the plate and sterilize the loop.

10. Rotate your plate 90º

11. Streak once through the second section and pull into the third section. Continue to streak the rest of the third section.

12. Put the lid back on the plate and sterilize the loop.

Isolating Bacteria Procedure

1. Divide one TSA, EMB, and MSA plate into 4 quadrants

2. Streak the quadrant in an “S” motion.

3. On the other TSA, EMB, and MSA plate, take the unknown mixed culture and perform a T-streak.

4. Tape their plates together and invert agar plates for incubation.

5. Incubate plates at 37°C for 24 hours.

Simmon’s Citrate and Urea Slants Procedure

1. Inoculate the slant tubes in an “S” like motion from the bottom of the slant to the top with an inoculating loop. The loop should be parallel with the slant.

2. Inoculate one slant with Escherichia coli and the other slant with Enterobacter aerogenes.

3. Tighten the screw cap on test tube, then loosen it by a quarter turn to allow oxygen to enter the test tube.

4. Incubate at 37˚C for 24 hours.