Microbiology lab test 3

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What are lysozymes?

antimicrobial enzymes that degrade the beta 1-4 bond between amino sugar molecules in peptidoglycan. Found in human tears and saliva and also egg whites of hen's eggs.


What do lysozymes do to bacteria?

Weakens the cell walls. As a result the solute pressure of the cytoplasm can cause the cell membrane to rupture resulting in cell lysis and death. Gram neg bacteria usually more resistant to lysozymes because of outer membrane.


Why is it important to test bacterial susceptibility to antibiotics?

to know which antibiotics are most effective in treating the bacteria.


define antimicrobial

compounds that kill or inhibit microbes


define antibiotics

antimicrobials produced by microbes that kiss or inhibit other microbes


know how to measure zones of inhibition

measurement of the diameter around the disc in mm to determine the microbes susceptibility to the antibiotic.


Kirby-Bauer method

  1. swab the entire surface of the plate with the organism to be tested.
  2. allow the agar surface to dry.
  3. either use an automatic dispenser or you can use forceps to place the antibiotic discs onto the medium.
  4. invert and incubate for 18 hours.
  5. measure the zones of inhibition in mm.
  6. Significance of zones determined by Kirby-Bauer chart.

What species of bacteria would you try to identify using the Enterotube?



Are Enterobacteriaceae gram negative or gram positive?

gram negative


are enterobacteriaceae oxidase positive or negative?

oxidase negative with only a few exeptions


In the reaction chambers that contain wax, what does it mean when the wax is separated from the agar?

the production of gas


The enterotube II procedure

  1. Remove organism from well isolated colony
  2. inoculate each compartment by first twisting the wire and then withdrawing it all the way out through the 12 compartments, using a turning movement.
  3. Reinsert the wire (without sterilizing), using a turning motion through all 12 compartments until the notch on the wire is aligned with the opening of the tube.
  4. break wire at notch by bending.
  5. Punch holes with borken off part of wire through the thin plastic covering over the last 8 compartments. Replace caps and incubate for 18-24 hours.
  6. after interpreting and recording positive results on the sides of the tube, perform the indole test by injecting 2 drops of Kovac's reagent into indole compartment
  7. perform the Voges-Proskauer test if needed for confirmation.

Name 3 bacteria often found in urine

  1. Escherichia coli
  2. Pseudomonas aeruginosa
  3. Staphylococcus species

how much bacteria must be present in a urine sample to indicate a urinary tract infection?

more than 100,000 CFU


how to calculate CFU/mL



uti testing

  1. collect urine sample
  2. transfer to sterile shake bottle and shake
  3. using calibrated inoculating loops .01 and .001 ml of urine are dispensed to liquefied TSA pours
  4. after completly mixing TSA pours between palms, content is poured into petri plates and cooled.
  5. After incubation colonies on the 2 plates are counted

Why is it important to perform antibiotic susceptibility testing on urine sample from UTI patient?

to make sure the antibiotic being used is effective.


Does toilet paper prevent our hands from being contaminated with bacteria?



We poured Escherichia coli onto a TSA plate and then wiped it with toilet paper, to represent what?



In what two ways do the Staphylococci differ from the Streptococci?

  1. Streptococci occur in chains rather than clusters
  2. lack the enzyme catalase

general characteristics of Staphylococci?

  1. Possess enzyme catalase
  2. can cause mixed infections with Streptococcus
  3. Pyogenic gram positive cocci
  4. responsible for nosocomial infections
  5. developed resistance to antibiotics
  6. exhibits hemolytic capabilities

What is pyogenic?

pus forming


What does it mean to be coagulase positive?

any degree of coagulation from a loose clot suspended in plasma to a solid immovable clot is considered positive.


Coagulase test

  1. With a wire loop inoculate plasma tube with organisms. use several loop fulls
  2. place tubes in 37 degree C water bath
  3. check for soidification of the plasma every 30 minutes

Coagulase positive example

Staphylococcus aureus


Coagulase negative example

Stahpylococcus epidermidis


Know three medically important species of staphylococcus

  1. Staphylococcus aureus
  2. Staphylococcus epidermidis
  3. Staphylococcus saprophyticus

What is tooth decay and how does it occur?

tooth decay is the break down of enamel and dentin. occurs in presence of high levels of sugar.


Name the bacterium associated with tooth decay?

Streptococcus mutans


Snyder Caries susceptibility test procedure

  1. collect saliva
  2. add .2 ml to liquified snyder test agar
  3. mix saliva and medium
  4. incubate for 24-72 hours
  5. green = negative
  6. yellow = positive

General characteristics of streptococci?

  1. part of the normal flora in our body
  2. gram positive cocci in pairs or chains
  3. facultative anaerobes (makes ATP w/o oxygen)
  4. separated into groups by hemolytic pattern and serotype

What is Alpha hemolysis

  1. partial helomysis
  2. shows greenish and faint halo around colonies
  3. discoloration due to loss of potassium from RBCs

What is Beta hemolysis

  1. complete hemolysis
  2. clear distinct halo around colonies

gamma hemolysis

no hemolysis


name a beta Strep species

Streptococcus pyogenes


name a alpha strep species

Streptococcus pneumoniae


How are Streptococci and Staphylococci classified?

  1. hemolytic patterns
  2. serotypes

Serological agglutination assays/tests use latex beads bound with what

antibodies specific for protein A


What is a positive and negative reaction

  1. positive = clumpin
  2. negative = no clumping