Essay: Introductory Microbiology Laboratory

UNKNOWN LAB REPORT
UNKNOWN BROTH #5
INTRODUCTION
There are microorganisms everywhere, meaning they are ubiquitous. Many of these microbes are nonpathogenic, even helpful. However, there are many that are disease causing pathogens. It is important to identify which microorganism is causing a disease before treating a patient. Identification of the specific microbe determines the appropriate drugs and treatment to combat the disease in the patient. The methods and techniques used to identify the unknown organisms have been acquired and applied from the microbiology lab class as well as from different sources including Microbugz (austincc.edu) and the Microbiology Openstax textbook.
MATERIALS AND METHODS
An unknown broth mixture labeled #5 containing both a gram-positive bacterium and gram-negative bacteria was handed out by the instructor.
The first procedure to be completed was to isolate the unknown bacterium on Trypticase Soy agar (TSA), Brain Heart Infusion (BHI) and Chocolate agar to obtain pure culture. This was accomplished by using the standard four quadrant streak plate method as described in Lab 4, Aseptic Technique and Streak Plates, on all 3 plates. Using the single line streak, aseptic inoculation was performed on the following plates: Blood Agar, Extra-cellular Amylase (Starch), Mannitol Salt Agar (MSA), MacConkey and EMB plates. Streaking these additional plates was in error. The nutrient agars and remaining plates were then incubated at 37º C for approximately 72 hours.
On all 3 nutrient plates separate and individual colonies with distinguishing characteristics grew. Morphology of colonies were observed, results were recorded, and Gram stain was performed. The individual cultures showed the following results, Unknown #1: White/Opaque Gram-positive cocci and Unknown #2: Translucent/colorless Gram-negative rod (bacillus). The MSA plate which is selective for only gram-positive bacteria resulted in an isolated Gram-positive colony which was then transferred onto a TSA plate and a colony from Gram -negative colony was also transferred onto a TSA plate and incubated at 37º C for 96 hours to promote growth of the pure culture.
After gram stains were completed, specific biochemical tests were performed for each gram-positive and gram-negative bacterium following the given flow charts for each.
 
Gram-positive bacteria tests performed:
1. Mannitol Salt Agar (MSA)
2. Vogues-Proskauer (V-P)
Gram-negative bacteria tests performed:
1. Lactose Fermentation done on the MacConkey Agar plate
2. H2S done on the SIM medium
3. Indole
RESULTS
Unknown #5 was isolated by the streak method on TSA, BHI, and Chocolate nutrient plates. Two colonies grew on those mediums and were gram stained.
The first colony had the following morphology on TSA, BHI, and Chocolate plates: 1-3 mm circular opaque white colored colony. The gram staining determined gram-positive cocci. This was inoculated onto different mediums. Table-1 lists all the biochemical tests, their purpose, reagents, observations, and results.
The second colony had the following morphology on TSA, BHI, and Chocolate plates: 3-4 mm irregular translucent colorless colony. The gram staining determined a gram-negative rod. This was inoculated onto different mediums. Table-2 lists all the biochemical tests, their purpose, reagents, observations, and results.
 
Table-1: Gram Positive Biochemical Test Results
TEST PURPOSE REAGENTS OBSERVATIONS RESULTS
GRAM STAIN To determine the Gram reaction of the bacterium Crystal Violet, Iodine, Alcohol and Safranin Purple Cocci clusters Gram positive cocci
MANNITOL
(MSA) To determine if the bacterium is able to ferment Mannitol None Growth, plate medium remained dark pink Halophile, negative Mannitol fermentation
VOGUES-
PROSKAUER
(V-P) TEST To determine if the bacterium will produce Acetoin V-P Reagent A (napthol), V-P Reagent B (KOH) Color change to Red Positive for Acetoin, Positive for utilization of the butlene glycol pathway
 
Table-2: Gram Negative Biochemical Test Results
TEST PURPOSE REAGENTS OBSERVATIONS RESULTS
GRAM STAIN To determine the Gram stain reaction of the bacterium Crystal Violet, Iodine, Alcohol and Safanin Pink rods Gram negative bacillus
LACTOSE FERMENTAION (MACCONKEY) To determine if the bacterium is able to ferment Lactose None Colorless growth Negative for Lactose fermentation
H2S (SIM) To determine if the bacterium is able to reduce Sulfur None Color change from clear to black, turbidity Positive for Sulfur reduction, positive for motile
INDOLE To determine the ability of an organism to hydrolyze tryptophan Kovak Color change to red Positive for Indole
 
UNKNOWN #5 GRAM POSITIVE FLOW CHART
Gram Stain
Gram positive cocci
Micrococcus spp.
Staphylococcus spp.
Streptococcus spp.
Enterococcus spp.
Mannitol Fermentation (MSA)
Growth
Staphylococcus spp.
Micrococcus spp.
No Fermentation
Staphylococcus epidermidis
Micrococcus luteus
Vogues-Proskauer (V-P) Test
Positive
Staphylococcus epidermidis
Unknown #5 Gram Positive Cocci – Staphylococcus epidermidis
 
INKNOWN #5 GRAM NEGATIVE FLOW CHART
Gram Stain
Gram negative rod
Enterobacteriaceae spp Escherichia spp.
Citrobacter spp Proteus spp.
Salmonella spp. Shigella spp.
Serratia spp. Pseudomonas spp.
Lactose Fermentation (MacConkey)
No Fermentation
Proteus spp.
Salmonella spp.
Shigella spp.
Serratia spp.
Pseudomonas spp.
H2S (SIM Medium)
Positive
Proteus spp.
Salmonella spp.
Indole
Positive
Proteus vulgaris
Unknown #5 Gram Negative Rod – Proteus vulgaris
Discussion/Conclusion
After following proper isolation techniques and performing several biochemical tests following Flow Chart # 3 on Gram-positive bacterium, it has been established that Unknown #5 Gram-positive cocci was Staphylococcus epidermidis. A Gram stain was performed on the bacterium which are positive cocci. A MSA plate was inoculated to detect growth and if the bacteria fermented mannitol. The growth eliminated Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus salivarius and Enterococcus faecalis. The growth indicated Staphylococcus. The lack of fermentation eliminated Staphylococcus aureus. A MR-VP broth tube was inoculated to run a V-P test. The V-P Reagent A (napthol) and reagent B (KOH) was added to the inoculated medium. Acetoin was produced clarifying that the Gram-positive unknown was Staphylococcus epidermidis.
Staphylococcus epidermidis is a commensalism resident flora using the dead skin cells of humans as nutrients. Most of the time it is avirulent, however it is an opportunistic pathogen especially in those who may be immunocompromised. It is community acquired, however it is also responsible for most nosocomial infections due to the biofilm growth on medical equipment such as catheters, implants or other medical equipment used in a medical/surgical setting. S. epidermidis is becoming increasingly difficult to treat due to multi-drug resistance.
After following proper isolation techniques and performing several biochemical tests following Flow Chart #4 on Gram-negative bacterium, it has been established that Unknown #5 Gram-negative rod was Proteus vulgaris. A Gram stain was performed on the bacterium which are negative rod. A MacConkey Agar plate was inoculated to determine if the bacterium fermented lactose. The negative lactose fermentation results eliminated Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes. A SIM Medium was inoculated to perform a H2S test. The positive result for reduction of sulfur eliminated Shigella flexneri, Serratia marcescens, and Pseudomas aeruginosa. The Kovac’s regent was added to the SIM Medium, a positive result for Indole was produced clarifying that the Gram-negative unknown was Proteus vulgaris.
Proteus vulgaris is a resident flora where most of the time is avirulent, however it is opportunistic pathogen. It can also be found in polluted water, soil, raw meat or in gastrointestinal tracts. It can cause urinary tract infections especially in women due to proximity to anus to the urethra and is responsible for many nosocomial diseases due to biofilm growth on catheters and on other medical devices. P. vulgaris is becoming increasingly difficult to treat due to multidrug-resistance.
During the lab exercises in finding the unknowns all of the tests ran exactly how they were supposed to. The initial and only problem encountered was inoculating too many plates with the combined Gram-positive and Gram-negative broth. This gave many unreliable results because both organisms were present. I was able to discard those unnecessary plates and clear up any confusing and unreliable data.
References
1. Microbiology Openstax Textbook 2016 (Textbook used in Introductory Microbiology course at Chattahoochee Technical College)
2. Dr. Nicole Bostick; D.C. Introductory Microbiology Lab Exercise Handouts, Chattahoochee Technical College, 2018.
3. aclsstlouis.com/5495/staphylococcus-epidermidis-microbiology
4. web.uconn.edu
5. merckmanuals.com/professional/infectious-diseases
6. austincc.edu/microbugz