Identifying Unknown Bacteria Lab

Report for “Unknown” Identification Project Jessica OlivaresFrancisco Alarcon-Chaidez12/02/2015

Negative and Positive IdentificationOf Unknown Bacterial Strains Report

Introduction/Purpose

The objective of this lab was to classify the unknown bacterial strains provided utilizing a number of biochemical procedures. In this lab each student was assigned two different strands of bacteria, one strand being a Gram negative bacteria and the other a Gram positive bacteria. Classifying and knowing the bacteria we had was done through a series of biochemical test. This lab project took 2 weeks of lab experimentation. Depending on each test the unknown strain transitioned to another test whether it was a positive result or a negative result we would go from there to another test and soon to finally identifying both of our bacteria’s. By following the procedures and methods of inoculation the results I obtained showed that my unknown strains were positive bacteria enterococcus faecalis, and negative bacteria Salmonella typhimurium.

Materials Types of media used included broth tube of mixed culture unknown bacteria strain #92. Used selective and differential media, for our unknown bacteria, such as slants, broth, and semisolid agar plates to identify negative and positive bacteria. To describe the morphology such as the growth, color, texture, and shape of our unknown strain we inoculated our bacteria into a differential media grown on a TSA plate. We used selective media MacConkey Agar and Mannitol Salt Agar for isolating and inhibit growth of the gram negative bacteria and gram positive bacteria. MacConkey agar is used for the isolation of gram-negative bacteria and variates lactose fermenting from lactose non-fermenting gram-negative bacteria we also used the gram staining technique to view the selected bacteria under the microscope. Catalase test was used to identify catalase negative bacteria the results were facultative in the production of bubbles and bacteria that test negative do not require oxygen as a terminal electron acceptor which anaerobes only ferment. Likewise following this we used the oxidase test as well. Following this we also used Gram positive test to identify unknown such as the identification of Hemolysis, Bile Esculin, DNase and the salt broth test. Gram-negative bacteria have a very thin peptidoglycan layer that is in between an inner cell membrane and a bacterial outer membrane fallowing the gram positive test we also conducted many of the gram negative test used to identify the correct gram negative sample. Methods

The lab project took 2 weeks of observation and identification of our unknown strains which began from Tuesday 11/10/2015-Thursday 11/19/2015 and consisted of four days of working with the bacteria sample. On the first day each student picked up an unknown mixed broth tube culture that was selected to work for two weeks, my unknown bacteria was number 92. This

sample was then inoculated by streaking for the isolation on a TSA agar plate. We then put the inoculated plate media into the incubator for the next lab period.

On the second lab day we took our TSA plate and inoculated the TSA plate into two mini Nutrient Agar plates to store for next the following week, the same day we collected our results from our TSA agar plate and we streaked our growth of bacteria from our TSA plates to be used for the gram staining technique in order to isolate both of the organisms. We then had to inoculate the two species to identify if our bacteria’s were Gram negative or Gram positive because from there we would follow charts that were specific for Gram negative or Gram positive bacteria. Gram staining is a method of determining bacterial species into two groups, it distinguishes bacteria by the chemical and physical properties of their cell walls which detects the peptidoglycan, Gram positive results in a purple/blue color while a Gram negative results in a pink/red color. Gram staining takes roughly around 5-8 minutes if done correctly. After gram staining we also conducted the catalase test which used a small sample of our unknown bacteria this test for a catalase enzyme I added a drop of hydrogen peroxide and positive bubbling occurs to differentiate between staphylococcus which is catalase positive and streptococcus which is catalase negative, an oxidase test was also conducted the same day. After initial gram staining was done TSA plate was used to also streak MAC Agar plates and MSA plates to test for positive and negative bacteria. We then disposed the TSA plate and incubated the mini NA plates for the next lab period.

On day three we used are MAC and MSA bacterial growth results to gather the test needed to identify both the species. We used the identification report sheet to figure out what test we needed to conduct for the negative and positive bacteria. We again inoculated the media appropriately using the mini NA plates that were made from the last lab period. After everything had been properly followed and each test was conducted accordingly we then incubated all the test we needed and disposed the 2 NA plates because we no longer needed for the experiment.

On the fourth and last day of this lab project we gathered all of our results from the incubator and recorded them into our data sheets for proper identification. We then examined all of our biochemical test and filled out our report sheet. After, we cleaned up our entire work station and disposed all our media and we finished by using our identification chart to match our results with the identity of our microbe. Finally we checked with our professor to see if our results were correct.

Results and Conclusions

From my results I obtained my unknown sample #92 Enterococcus faecalis as my positive bacteria and Salmonella typhimurium as my negative bacteria. Below I have provide a data sheet that illustrates my observations which also shows the conducted test and results of both of my unknown species.

Table 1: The following table has a list of my data recording that were observed during the lab inoculation of the two species.

For identification of culture (morphology-growth, color, texture, shape. Etc.)

Unknown ID Report

Grown on TSA Completely filled with bacterial growth colonies cannot yet be distinguished which is gram positive or gram negative species.

Grown on MAC (inhibits negative bacteria)

Pink to red growth with or without bile precipitate, some colonies were barely pink.

Grown on MSA (inhibits positive bacteria)

Yellow growth and halo showed up as positive bacteria.

Viewed under microscope (gram stain)

Gram-positive bacteria species inoculated to identify if our bacteria’s were Gram negative or Gram positive bacteria. Chemical and physical properties of their cell walls detected the peptidoglycan, Gram positive results in a purple/blue color while a Gram negative results in a pink/red color.Gram-positive bacteria appeared a bit more red than pink Gram-negative bacteria appeared a really deep purple color

Table 2: By conducting the preliminary test I first streaked the media

on a glass slide. And did tconducted the gram stains for the species. The catalase test created no bubbles which ment ngative sample bacteria.

Table 2A: The Hemolysis test is where abacteria is inoculated onto blood agar which uses bacteria sample to test how much hemolysins the bacteriaproduces which is an exotoxin that destroys red blood cells (RBCs); and in the case of my bacteria it tested out gamma.

Figure 1: Shows the DNase sample of my inoculated bacteria. The DNase agar contains nutrients for the bacteria or DNA and uses methyl green as an indicator.

:

Preliminary Test

Gram Stain + -

Catalase + -

Oxidase + -

Gram Positive Test

Hemolysis α β γ

6.5% Salt Broth + -

Bile Esculin + -

DNase + -

Figure 2: Shows the hemolysis sample collected of my inoculated bacteria. The blood agar showed the growth of the bacteria which appears as a pink growth colonies. It showed the exotoxins that destroys the red blood cells.

Table 2B: Gram negative bacteria was inoculated and put into different vials that was isolated using differential media. The gram negative test identified the correct microbe.

Gram Negative Tests

TSI SLANT ALK ACIDBUTT ALK ACIDGAS + -H 2S + -

SIM H 2S + -INDOLE + -

MOTOLITY + -PR-GLUCOSE pH + -

GAS + -PR-SUCROSE pH + -

GAS + -PR-LACTOSE pH + -

GAS + -DECARBOXYLASE LYSINE + -

ORINTHINE + -MR + -VP + -

CITRATE + -UREASE + -

PHENYLALANINE DEAMINASE + -

Table 3: The proper identification was done by only using the TSI Slants, SIM medium. & The Decarboxylase.

Table 4: The following table was identified by conducting the appropriate gram stain results fallowed by the catalase test, oxidase test and the Hemolysis test.