Microbiology lab

Introduction

The isolation and cultivation of bacteria enable in combination with cultivation-independent methods and even the function of microbes. The isolation of a bacteria remains to be an essential step in the diagnosis and management of the illness. The steps involved in isolation are a collection of the specimen, preservation and transportation, microscopic examination of the sample, and the various methods used for isolation of the bacteria. On collection, the example will be sent for analysis with a suspected bacterial infection. Other infections would be very hazardous hence making them be treated with a high level of safety measures. Immediately specimens are collected, they are sent for preservation measures that are stored at 4 degrees, thus preventing bacterial multiplication. Later on, viewed on a microscope, an activity known as microscopy. In the laboratory, specimens are usually separated by different methods such as culture methods and non-culture methods. In non-culture methods, techniques such as PCR (Polymerase Chain Reaction) are used for the isolation and the identification of the bacteria. In culture methods, they can be grown on either: solid media, liquid media, or automated systems. On the interpretation of the results at the end of the processes, the key factors include the type of the specimen, type of the bacteria recovered, knowledge of the normal human flora, and even details of the antibiotic therapy.

Methods:

This bacteria was isolated by performing multiple rounds of isolation streaks. After it was isolated multiple tests were performed in order to identify the bacteria Gram staining was the first test performed. Gram staining is used to determine if a bacterium is gram positive or negative. 

A MSA test was performed in order to determine if the bacteria was salt tolerant and mannitol fermenting. MSA also tends to select for Gram positive bacteria, however some Gram negatives are able to grow on it as well. MSA is selective and differential. A positive reading is shown by the pink agar turning yellow.

The next test performed was a Macconkey Agar test. Macconkey Agar selects for gram negative bacteria by using bile salts and crystal violet. These elements create an environment that is too harsh for gram positive bacteria to grow. The Macconkey test is also differential for lactose fermentation. A positive result is indicated by the agar turning pink while for a negative result the agar remains yellow.

Blood Agar tests to see if the bacteria is capable of hemolysis. Hemolysis means the rupture or destruction of red blood cells. There are three different levels of hemolysis: Beta, Alpha, and Gamma. Beta indicates total breakdown of the blood. Alpha indicates partial breakdown while Gamma indicates no break down.

The bacteria was then streaked onto a DNase plate. This test is selective for the presence DNase which breaks down DNA. The plate changes from green to yellow when the enzyme is present.

The next test performed used SIM tubes. This tube tests for three different factors: Sulfide production, Indole formation, and motility. Blackening of the tube signifies the bacteria is positive for sulfide production. Spreading signifies that the bacteria is motile which suggests the bacteria have flagella or cilia. If the tube were to have a red ring at the tops this signifies the bacteria are positive for the indole test.

The Phenol Red Broth tests for carbohydrate fermentation. If the broth turns yellow this signifies that the bacteria is positive for carbohydrate fermentation. Phenol Red Broth also tests for gas production. This is determined by looking at the Durham’s tube. If fluid has left the Durham tube, this signifies that gas has been produced. 

The bacteria was also tested in anaerobic conditions to determine if the bacteria could grow without oxygen. 

Lastly the bacteria was tested to see if it would react with Hydrogen peroxide. This is called a Catalase test. If oxygen bubbles are created then this indicates a positive result.

Results:

After isolation and Gram staining the bacteria was observed under a microscope. The bacteria appeared to be purple in color. It appeared as circular units in a chain. This indicates streptococcus bacteria. The purple color created by the Gram staining indicates that the bacteria is gram positive.

The result of the MSA plate was a negative reading. This indicates that the bacteria is not salt tolerant or mannitol-fermenting. It also brings up conflicting results due to the fact that MSA selects for gram positive bacteria yet the agar did not turn yellow which indicates a negative result.

The unknown bacteria also showed a negative result for the Macconkey Agar test. The agar remained yellow. This indicates that the bacteria is not gram negative. It also indicates that the unknown bacteria does not fermentate lactose.

In regards to the Blood Agar test the bacteria showed no ability of hemolysis. It was determined to show Gamma levels which means no breakdown of red blood cells occurred on the plate.

For the Sim tube, the bacteria showed no sulfide production or indole formation. However, the tube did show signs that the bacteria is motile.

The unknown bacteria indicated negative for carbohydrate fermentation during the Phenol Red Broth Test. It did not show any signs of gas production either.

The unknown bacteria was found to not be able to grow in anaerobic conditions.

The unknown bacteria was found to be positive for the Catalase test. It produced oxygen bubbles in the presence of hydrogen peroxide.

According to the Gideon Microbiology search, based on the results of the various tests the unknown bacteria could be Arthrobacter agilis, Auritidibacter, or Rickettsiae.

Discussion:

The primary purpose of this study was to examine the function of microbes using cultivation and isolation techniques. Earlier research studies suggests that the growth and development of cultivated microorganisms plays an integral part of modern microbiological, biotechnical, and medical research (Cheptsov & Minaev, 2014). Without an expected hypothesis, the unknown bacterial examination led to some significantly interesting results. Based on observed features, the unknown bacteria appeared to be purple and shaped as a steptococcus. Furthermore, our gathered morphological features indicate that the unknown bacteria could be the following species: Proteus mirabilis, Providencia stuartii, or Serratia marcescens. Proteus mirabilis is a gram-negative bacterium that is commonly known for its ability to swarm across surfaces in an unually bulls’-eye pattern. Typically, this bacteria affects the urinary tract, especially in patients undergoing long term catheterization. Secondly, P. stuartii is also a gram negative bacillus. However, it is often found in soil, water, and sewage. alike P. mirabilis, this bacteria also causes urinary tract infections. P. stuartii is also considered to be a nosocomial pathogen in locations such as nursing homes and acute care hospitals. The same information pertains to the last bacterium, S. marcescens. However, S. marcescens is not only associated with urinary issues, but also respiratory infections, endocarditis, osteomyelitis, septicemia, wound infections, eyes infections, and meningitis. All of these bacterias have been found to have caused serious health infections from the lack of hand washing. Based on the collected infromation about the unknown microbial bacteria, it is likely to be associated with either P. mirabilis, P. stuartii, or S. marcescens.