Microbiology Log
Day 1: Tuesday April 15
A. Enumeration of a urine sample:
In order to determine the number of organisms per ml in a urine sample, the first thing that has to be done is a dilution of the original sample. The following dilution was carried out: 1ml of the urine sample was added to 99ml of water which created the 10^-2 dilution. From this sample, 1ml was taken out and added to a 9ml water blank creating a 10^-3 dilution and then one last time a 1ml sample was taken out of this sample and added again to a 9ml water blank giving a final dilution of 10^-4.
To count the number of bacteria in these dilutions two methods were used; 1ml of each sample was placed in a petri dish and then TSA was poured over the samples for a standard pour-plate count procedure. The second method utilized the Petrifilm Aerobic count method, which takes 1ml of each sample and is placed underneath the top film. The film is then placed down onto the sample and then the sample is distributed evenly with a plastic spreader that is pressed down onto the petrifilm recessed side down in order to create a circle of the sample that is solidified. Plates and petrifilm were place in the 37degree room till next period.
B. Isolation and Identification of all M/O’s
My unknown number obtained was #7. A gram stain was performed of the mixture and I saw the following organisms: one gram positive cocci in clusters, and two gram negative bacilli that I couldn’t differentiate.
The next step was to dilute the unknown mixture by adding a few drops of it to a 9ml water blank and then streaking this diluted mixture onto BAP, MAC, and EMB plates in order to obtain isolated colonies. All three organisms should grow on the BAP plate, but only the gram negatives should grow on the MAC and EMB plates since they have components (bile salts and crystal violet, methylene blue respectively) to inhibit gram positive organisms. These plates were then incubated at 37degrees, except the BAP plate which was placed in the CO2 chamber for 24hrs and then put into the cold room.
Day 2: Thursday April 17
Note: The day in between classes, plates (mainly BAP) were observed for possible S. pneumonia: since this organism was not present, they were placed in the cold room until Thursday.
A. Enumeration
Plates and petrifilm were counted. The 10^-2 diluted plate and petrifilm were TNTC and the 10^-4 plate and petrifilm were TFTC. Both the plate and petrifilm had a count of approximately 42 colonies on the 10^-3 samples. Counts between 25 and 250 are considered statistically significant. Therefore the urine sample has about 42,000 CFU/ml. This count indicates insignificant bacteriuria because it is not over 100,000 CFU/ml.
Pros and Cons of each method:
Standard Pour Plate Count:
This method is standard just like the name implies. It works and can be useful if there is no other method available. Cons for this method can be aspects such as: having to make sure the bacteria sample is mixed well with the agar, differentiate the different colonies when counting, hoping that the colonies actually grow, etc.
Petrifilm Aerobic Count:
I prefer this method over standard plating method because it’s quick and reliable. The counting is easier because the colonies are red and in the center of the petrifilm vs. trying to resolve between two colonies stuck together, identifying colonies at the bottom or edge of the plate, etc. The only con about this method that seems to be of any importance is the fact that it’s meant for aerobic organisms only.
B. Isolation and Identification
The plates went as follows:
BAP: there seemed to be gamma and some beta hemolysis. White, medium sized, raised colonies were very distinctive and these I decided to name as number 1. However, the two other colonies were hard to decipher and seemed grayish and white, and very small and clumped.
MAC: after 24hrs there appeared to be one colony type that was purple, but after time, the colonies turned clear and made the purple agar turn yellow. The gram negative appeared to be a lactose fermentor.
EMB: at first glance there seemed to be two types of colonies; a purple big colony (named #2) and pink big colonies (originally #3). However these became one type of colony after time and made me realize that my initial idea of there being two gram negative bacteria in my sample was probably wrong.
I gram stained three colonies I thought were different and grew the gram positive on TSA and the two gram negatives on KIA agar. The gram stains showed the same results as before. A gram positive cocci and two gram negative bacilli (turns out these two were the same organism), so I thought I had isolated my organisms correctly.
Day 3: Tuesday April 22
B. Isolation and Identification
My streaked plates were checked for technique.
In checking my KIA slants, the two gram negatives showed the exact same results: A/A with gas production so I knew that it was probably the same organism. With this knowledge I knew that my #2 organism was my gram negative bacilli probably from the Enterbacteriaceae family. My TSA slant grew my gram positive cocci, my #1, and so I had only one problem, what was my third organism? I searched over all my plates: the BAP plate was too hard to distinguish a colony besides my white one that I had already determined, the MAC plate only grew one gram negative which I also had determined; and in taking another glance at my EMB I saw a small pink colony hidden amongst my large gram negative, purple colonies. I gram stained this colony and it turned out to be gram positive; which should not have grown on an EMB plate. With that I was given a virtual organism for my #3.
Tests to be preformed:
Organism #1: Gram positive cocci (clusters)
TSA slant showed white streak, BAP showed white colonies and possible Beta hemolysis
Tests ordered: MSA plate, coagulase test, and catalase
Organism #2: Gram negative bacilli (random)
KIA: A/A, gas formation, BAP showed possible gamma hemolysis
Tests ordered: Citrate slant, LIA, MIO, and Urease
Organism #3: Virtual, Gram positive cocci (chains)
BAP plate showed gamma hemolysis
Tests ordered: Catalase and Bile esculin
Place in appropriate incubation areas.
C. Other Tests: API 20E and Enterotube
A purple colony from my EMB plate was used to inoculate an API-20E strip and an Enterotube. The organism should be gram negative bacilli.
The API-20:
The middle of a purple colony on my EMB plate was isolated and placed in a water sample to dilute the sample. Then samples were placed into each compartment on the API strip. The compartments with no lines were filled to the bottom line with the sample, the compartments underlined were filled to the bottom line with sample and then oil was added to fill the open oval, and the compartments boxed in with lines were filled to the top with sample.
Enterotube:
Each side had a cap and these were twisted off. The white side’s needle was inoculated and then the blue side pulled the entire needle thru to inoculate each test and then it was broken off. Holes were poked thru the tests that required oxygen.
Day 4: Thursday April 24
B. Isolation and Identification
Organism #1: Staphylococcus aureus
Gram Reaction
Gram positive
Purple color
The cell wall stayed intact due to the thick peptidoglycan layer and crystal violet was maintained.
Morphology
Cocci, clusters
BAP: Hemolysis
Beta
White colonies, yellow hemolysis
Complete lysis of RBCs.
MSA plate
Positive, yellow agar
Pink agar to yellow and growth
7.5% sodium chloride is hard to grow on. Selective for staphylococci, and ferments mannitol.
Coagulase
Positive
White clumping
Bound/free coagulase is attached to the cell wall and reacts with fibrinogen in plasma.
Catalase
Positive
White compound; fizzes
Catalase present. Hydrogen peroxide is converted into water and oxygen gas.
Organism #2: Enterobacter aerogenes
Gram Reaction
Gram negative
Red color
Thin peptidoglycan layer and crystal violet was not maintained therefore secondary stain is present.
Morphology
Bacilli, random
BAP: Hemolysis
Gamma
Grayish colonies
No lysis of RBCs. No change in medium except for growth.
KIA
A/A, gas formed
All yellow, air bubbles
Lactose and Glucose were both fermented.
Citrate Slant
Positive
Green to Blue
Can use citrate as sole carbon source.
LIA
Positive, K/K
All purple
Lysine decarboxylase is present. Lysine deaminase is not present.
MIO
Positive, Negative, Positive
Cloudy, stayed clear after Kovac’s reagent, purple color
Is motile, no indole formation, lysine decarboxylation is present.
Urease medium
Negative
No color change
Does not hydrolyze urea.
EMB/MAC
Purple/Purple & clear
Purple colonies/clear
Lactose fermenter
Organism #3: Enterococcus faecalis
Gram Reaction
Gram positive
Purple color
The cell wall stayed intact due to the thick peptidoglycan layer and crystal violet was maintained.
Morphology
Cocci, chains
BAP: Hemolysis
Gamma
Grayish/white colonies
No lysis of RBCs. No change in medium except for growth.
Catalase
Negative
No change.
No catalase present. Hydrogen peroxide is not converted into water and oxygen gas.
Bile esculin
Positive
Slant turned black.
Hydrolysis of Esculin occurred in the presence of bile.
C. Other Tests
API-20E results: see paper
Number: 430 5773
Classified: Enterobacter aerogenes
Enterotube results: see paper
Number: 36361
Classified: Enterobacter aerogenes
D. Another method to identify Enterococcus faecalis:
Enterococcus faecalis is very antibiotic resistant whether naturally or through R-plasmid transmission. Vancomycin is an example of an antibiotic that it could possibly be resistant to. In this case, other tests that can be performed to distinguish this bacterium can include antibiotic disks inhibiting of growth.
Reference: http://web.mst.edu/~microbio/BIO221_2005/E_faecalis.htm
