ID of Gram Negatives Flashcards
Describe the difference between aerobes, anaerobes, facultative anaerobe, capnophilic and microaerophilic
Aerobes: grow in the presence of oxygen
Anaerobes: grows in the absence of oxygen, oxygen is usually toxic to anaerobes
Facultative anaerobe: can grow aerobically or anaerobically
Capnophilic: requires CO2 for growth
Microaerophilic: grows in a low concentration of oxygen but not in its absence or in ambient air
Gram negative bacteria are often more drug resistant than gram positive bacteria, why is this so?
Gram negatives have a more complicated cell wall which is an effective barrier against some antimicrobials
What are the common characteristics of the enterobacteriaceae family?
Gram negative rods Oxidase negative Ferment glucose Facultative anaerobes Reduce nitrate to nitrite
What is the only enterobacteriaceae that is oxidase positive?
Plesiomonas
What is MacConkey agar?
A selective and differential growth medium used to isolate gram negative bacteria. It inhibits growth of gram positives by containing bile salts and crystal violet. It contains lactose to differentiate lactose fermenting enterobacteriaceae from lactose non-fermenting enterobacteriaceae.
Describe the nitrate test
Tests gram negatives for their ability ability to reduce nitrate to nitrite (has nitrate reductase) as well as the ability to perform nitrification on nitrate or nitrite to make molecular nitrogen. Incubate potassium nitrate broth with bacteria. Add alpha naphthylamide and sulfanilic acid. If nitrate has been reduced to nitrite, the broth will turn red. If the broth doesn’t turn red, it means that either the organism cant reduce nitrate or it was able to nitrify nitrate to nitrogen or ammonia. If broth doesn’t turn red, add zinc powder. If nitrate is present, the zinc will reduce it to nitrite and cause the broth to turn red: this means the bacteria CANNOT reduce nitrate. If there is no colour change after addition of zinc powder, there was no nitrate present to reduce and all of it was reduced to either ammonia or nitrgogen
Describe how a TSI slant test works
It is a test used to differentiate enterobacteriaceae based upon their ability to reduce sulfur and ferment different carbohydrates. TSI agar is a differential medium containing 3 sugars: glucose (1%), lactose (10%) and sucrose (10%), ferrous sulfate and phenol red. Inoculate broth and incubate for 10 hours. Glucose is the limiting sugar and will be depleted first. If the bacteria can ferment glucose and at least one of the other sugars, the slant will remain yellow (indicating acid made by sugar fermentation). If the bacteria can only ferment glucose, it will metabolize the amino acids next. AA metabolism requires oxygen, will only occur in the aerobic slant and creates an alkaline environment, turning the slant agar red. If the bacteria can reduce sulfur, the hydrogen sulfide produced by the reaction will react with the iron to form iron sulfide, a black precipitate. Sulfur reduction requires an acidic environment, so if it occurs, some sugar fermentation took place.
You inoculated 5 TSI slant tubes with different enterics and incubated them for 10 hours. After the 10 hours, the results of the tubes were as follows: 1) Acid slant/ acid butt, 2) alkaline slant/ acidic butt, 3) alkaline slant/alkaline butt, 4) alkaline slant/black butt 5) acidic slant/black butt with gas. What was the unknown bacteria able to metabolize in each tube?
1) Glucose fermented as well as lactose and/or sucrose
2) Glucose fermented but neither lactose nor sucrose fermented
3) Afermenter: none of the sugars fermented, metabolized amino acids only
4) Glucose fermented only, amino acids metabolized, sulfur reduced
5) Glucose and lactose and/or sucrose fermented, sulfur reduced, gas produced
Describe the general characteristics of vibrios and aeromonas with respect to their similarities to and difference from enterobacteriaceae.
Primarily found in water sources. Gram negative, facultative anaerobes that ferment glucose like enterobacteriaceae. Unlike most enterobacteriaceae, they are oxidase positive.
What are the 3 key tests you would use to test if an unknown gram negative bacteria was a vibrio spp.?
1) Salt tolerance: vibrios can grow in up 6% NaCl
2) Oxidase: vibrios are oxidase positive, most enterobacteriaceae are not
3) Vibriostatic agent O129: most vibrio spp. are susceptible to O129 whereas most other bacteria are resistant
What is TCBS agar used for? What does is contain?
Used as a highly selective medium for Vibrios.
Oxgall: inhibits growth of gram positives
Sodium thiosulfate/ferric chloride: detection of sulfur reduction, inhibits growth of enterobacteriaceae
Sucrose
Vibrios grow as yellow colonies
You have isolated a gram negative cocco-bacilli from a nasopharyngeal swab. It is oxidase positive, facultatively anaerobic but grows well in CO2 and doesn’t grow on MacConkey agar. What bacteria would you guess it is? What test could perform to confirm it is identity?
Haemophilus influenzae. Perform the porphyrin test.
Describe how to perform the porphyrin test and interpret the results. What clinically important bacteria would give a negative test?
Test used to determine and isolate’s X factor (protoporphyrin) requirement. Make a heavy suspension of the organism in aminolevulinic acid and incubate for 4 hours. After 4 hours, observe the suspension under UV light. Positive test: red fluorescence, indicates the organism converted aminolevulinic acid to porphyrins and is therefore X factor independent. Negative test: no fluorescence, organism requires X factor for growth. Haemophilus influenzae gives a negative porphyrin test because it cannot make poryphyrins on its own.
Haemophilus is able to grow on chocolate agar but not on SBA? Why is this so? Sometimes when growing S. aureus on SBA, satelitte colonies of H influenzae may be observed, why is this so?
Haemophilus can’t lyse red blood cells to obtain heme for growth, chocolate agar has red blood cells lysed during preparation and thus the heme is available to the organism. Haemophilus can satellite off the beta haemolytic S aureus and obtain the heme
Describe the basic morphology an characteristics of afermenters. Remember that there is no family designation for them.
By definition they do not ferment glucose. Most are gram negative bacilli or cocco-bacilli. Most are obligate aerobes and grow best at 35 degrees. Often oxidase positive. May not grow on MacConkey.
