Unknown Bacteria Sample #15 Lab Report

Sanzida Taslim
Abstract
The goal of this lab experiment was to identify an unknown bacteria sample using different
selective and differential media. The different agar plates were streaked with this unknown
bacteria and left to incubate for 24 hours. The results from the growth of the bacteria on the agar
plates show that there was contamination of the plates during some part of the procedure. The
carrying results suggested that the unknown bacteria was possibly salmonella, and was
contaminated with acid producing B. Meg bacteria. One plate, the Brilliant Green Agar, was an
outlier from this hypothesis, but picture results suggests that there may have been an error when
this plate was being prepared, which caused it to produce the no growth results.
Introduction
The objective of this lab experiment is to identify an unknown bacteria sample using the
different agar plates, filled with selective and differential media. Selective media is media that
contains certain ingredients or conditions that only allow for the growth of particular
physiological types of microbes. Differential media is media which contains ingredients that
allows differentiation between different bacterial types based on the colony morphology or the
reactions produced by the bacteria in the medium. There is medium that can be described as both
selective and differential.
For example, Mannitol Salt Agar contains 7.5% salt, which inhibits many non-halophilic
organisms. This plate is used as a selective medium for Staphylococci, which can withstand these
conditions. It also contains mannitol, a sugar staphylococci can use to produce fermentation. Its
pH indicator is Phenol Red, which changes to yellow in the presence of acid, which show lactic
acid produced during fermentation, therefore differentiating fermenting bacteria and nonfermenting bacteria.
Eosin-Methylene Blue agar plate allows differentiation between enteric lactose fermenters and
non-fermenters. It is also used to identify E. Coli, due to the fact that this bacteria produces a
characteristic blue-black color with a metallic green sheen. Pink and mucoid growth, or dark

(purple to black) growth show possible coliform bacteria formation. Pink growth is evident of
little acid production and dark shows a lot of acid production.
Vogel Johnson agar plates can be considered as both selective and differential medium. It can
detect S. aureus, which is identified the coagulase-positive and mannitol-fermenting strains. It
contains tellurite, lithium chloride, and high glycine concentration for inhibition of organisms. It
can also detect staphylococci carriers.
Hektoen Agar Plates can also be considered a differential and selective media. It contains bile
salts, which inhibit gram positive bacteria. It produces greenish colonies with black centers if the
bacterium grown is Salmonella, and produces bright orange colonies when Enterobacter is
produced.
The Blood Agar Pate is a plate of differential medium. It is enriched with sheep blood, and is
used to see growth of bacteria and observe different types of hemolysis caused by growing
bacteria. Alpha hemolysis, partial decomposition, is evident with green discoloration. Beta
hemolysis, complete decomposition, is seen with clearing around the colony. Gamma hemolysis,
no decomposition, shows no clearing or discoloration, but growth can be seen.
The Brilliant Green Agar Plate is a plate of selective medium. It produces growth of many
Salmonella species. Growth of all gram-positive and most gram negative bacteria is inhibited. It
also shows differentiation between lactose fermenters, which could be E. coli and non-lactose
fermenters, Salmonella. Yellow growth shows evidence on lactose fermenting bacteria, while
dark red shows non-lactose fermenting bacteria.
MacConkey agar plate uses crystal violet to inhibit the growth of Gram Positive Bacteria, and
permits the growth of Gram Negative Bacteria, also containing sugar lactose and Bile salts. Its
pH indicator is neutral red. This agar differentiates bacteria on their ability to ferment lactose
sugar as well. Lactose fermenting bacterium produces pinkish colonies, while non-fermenting
bacteria produces purple colonies.
The Tryptic Soy Agar Plate is a standard plate which will be used as a control medium to
compare the other plates to.
Materials

All of the materials that were used for this procedure are listed below:
-unknown bacteria sample given in liquid tube
-blood agar plate
-brilliant green agar plate
-MacConkey agar plate
-Tryptic Soy Agar Plate
-Hektoen Enteric agar Plate
-Vogel and Johnson agar Plate
-Mannitol Salt Agar Plate
-Eosin Methylene Blue agar Plate
- Loop Applicators

Methods
The methodology that was exercised for this lab procedure was the streak plate technique on the
various selective and differential media that we identified in class.
We were first given unknown samples of bacteria, which were all individually numbered, and
given the differential plates. Using a fresh loop applicator every time, we streaked each plate by
dipping the applicator into the sample and spread it over the agar on each plate, (which were
placed only slightly ajar and immediately capped after application of unknown) until all plates
were streaked with the unknown bacteria. These plates, which were labeled beforehand, were put
away and stored in an incubator for 24 hours. After this time period, results were observed and
recorded.

5. Results (4pts)

Figure 1: Blood Agar Plate

Slight green discoloration shows that alpha hemolysis, or partial decomposition, has occurred.
(this may suggest that the bacteria could possibly be B. med or Strep. P)

Figure 2: Brilliant Green Agar Plate

The brilliant green agar plate shows very poor to no growth at all. This rules out salmonella, and
possibly E. Coli

Figure 3: MacConkey Agar Plate

This plate shows clear, translucent colorless colony growth, suggesting that whatever bacteria
that have grown on this plate did not form any lactic acid, making it non-fermenting bacteria.

Figure 4: Hektoen Enteric Agar Plate

This plate shows some blackish green formation where the unknown was streaked.

Figure 5: Vogel Johnson Agar Plate

This plate shows no growth at all, ruling out the possibilities of the bacteria being S. aureus, B.
Meg, and Staph. A, which would have shown different results.

Figure 6. Mannitol Salt Agar

The strong yellow colonies suggest that the bacterium does produce an acidic byproduct due to
the fact that it changed the color of the pH indicator in the Mannitol Salt Agar. Also, looking
solely at this plate, this suggests that the bacteria can possibly be B. meg or Staph A.

Figure 7: Eosin-Methylene Blue Agar Plate

This agar plate shows translucent/amber colonies, which suggest that the bacteria can possibly be
salmonella, which does not produce lactose.

Figure 8. TSA Plate, control, basic medium

Agar Plate
Blood Agar

Results
Alpha Hemolysis

Possible Grown Bacteria

-Strep. P

Brilliant Green Agar

MacConkey Agar

No Growth
Colorless Translucent

-B.Meg
-Not Salmonella, possibly not E.Coli
-Salmonella

Hectoen Enteric
Vogel Johnson
Mannitol Salt Agar

Colonies
Blackish Green formation
No Growth
Yellow Colonies

-Salmonella
-Not B. Meg, Staph. A, or M. Smeg
-Acid producing Staph A.

Translucent/Amber

-Acid producing B. Meg

-Salmonella

EMB Agar

Colonies
Table 1: Results Chart
Discussion
The results that came from the differential and selective agar plates were incredibly inflicting
with one another, suggesting that the agar plates were contaminated during or after the procedure
were done. For example, the Salt-mannitol agar clearly shows evidence of an acid producing
bacteria, but many other plates suggest the growth of Salmonella, which is a non-lactose
producing bacteria. I believe that the plates were probably contaminated and in contact with Acid
producing B. Meg bacteria, as some plates suggest the growth of B. Meg. The Vogel Johnson
plate was probably not contaminated, which is the reason as to why it did not show any growth.
Because many of the plates suggest that Salmonella was produced, my guess is that the original
unknown bacterium was Salmonella. The Brilliant Green Agar results do conflict this conclusion;
however, when observing the pictured results, the plates seems to be absolutely empty, with no
evidence of any original smearing, unlike the other plates, which may suggest that due to some
human error on my part, this plate did not properly have the unknown applied.