Unknowns Project - Micro, Papers of Nursing

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Unknowns Project

Authors: Breanna Burton Date: June 29, 2025 Lab Day & Time: Thursday @ 0900

Introduction

The purpose of this project is to identify two unknown microorganisms. There are many reasons for understanding and being able to identify these microorganisms, in some cases, it can not only help us understand what is causing the infection we are also able to treat it properly. Identifying microorganisms and understanding how they grow and are broken down can assist in the creation of effective antibiotics. This experiment was executed by following a series of experiments mapped out with the knowledge gained in the microbiology laboratory class for identifying microorganisms.

Materials and Methods (Daily Log)

November 3, 2023

At the beginning of this project, two unknown microorganisms were collected in inoculation test tubes. For this particular lab report, the two unknown microorganisms are labeled #64 and #68. The knowledge that has been gained so far in the previous laboratory settings has provided the route to be taken in identifying the two unknown microbes. The steps that were taken for each experiment were through the use of the Microbiology lab manual by Leboffe and Pierce. (Leboffe and Pierce) The two unknown microorganisms were collected and the first step of identifying the microbes was to create a streak plate. The quadrant streak was performed for the unknown microbe #64 and #68. By using a wire

November 7, 2023

After the observations of the new streak plates labeled #64A and #64B, they were isolated correctly and there appeared to be more than 30 isolated colonies for both plates. On the same day, the Catalase test was conducted per the microbiology lab manual (Leboffe and Pierce). Each bacteria was smeared onto separate slides and hydrogen peroxide (H 2 O2) drops were added to each bacteria, the drops converts the H 2 O 2 to water and oxygen gas when combined with the bacteria. This is an important test to distinguish if the unknown bacterias are staphylococci or streptococci.

Seeing that the catalase test for slides #64A and #64B produced bubbles, the map that was created before the project (attached at the bottom) shows that a Glucose test is now required. A small sample of bacteria #64A was obtained from the nutrient streak plate and placed into a test tube that contained glucose phenol red fermentation broth; the same was done for bacteria #64B. Both test tubes were placed into the inoculator set to 30° C, they were left in the inoculator for 24 hours. This test was conducted per the microbiology lab manual. (Leboffe and Pierce) As for the bacteria labeled #68, there were no bubbles produced, the flow chart indicates that a Mannitol test is now required. A small sample of bacteria obtained from the streak plate labeled #68 was placed into a test tube that contained mannitol phenol red fermentation broth; and then placed into the incubator set at 30° C, it was left in the incubator for 24 hours.

November 8th, 2023

The glucose and mannitol test tubes were removed from the inoculator and results were observed. Both unknown bacteria in the glucose test tubes produced an acidic result (the solution turned yellow). This test shows the bacteria’s ability to ferment the sugar and the way a microbe metabolizes a carbohydrate such as glucose (dextrose). The unknown bacteria in the mannitol test tube also produced an acidic result (the solution turned yellow), this indicates that the bacteria can ferment the carbohydrate (sugar) mannitol as a carbon source.

The second part of this day for bacteria #64A Casein test was set up. A milk agar plate was obtained from the refrigerator and a small sample was obtained from the nutrient streak plate labeled #64A and the sample was rubbed onto the milk agar plate in a line. Then another small sample was taken off the nutrient streak plate #64A and stabbed into the milk agar plate, the stabs were repeated two more times for a total of three stabs into the milk agar plate. Then the milk agar plate was placed into the 30°C inoculator for 36 hours and then moved to the fridge. As for bacteria #64B a Nitrate Reductase test was set up. A small sample was pulled from the nutrient streak plate labeled #64B, the sample was placed inside the test tube with the Nitrate Reductase solution. After the test was completed the test tube was placed into the 30°C inoculator for 36 hours and then moved to the fridge. Both tests were conducted per the microbiology lab manual. (Leboffe and Pierce) The unknown bacteria #68 needed a growth streak plate set up. A small sample of the bacteria was obtained from the nutrient streak plate labeled #68 and a lawn technique was performed on another nutrient plate. After the plate was set up, it was then placed in the incubator set at 45°C for 36 hours and then moved to the fridge, to see if the bacteria could grow in warmer conditions. The test was conducted per the microbiology lab manual. (Leboffe and Pierce)

November 17

th

The Milk agar plate for unknown bacteria #64A was removed from the fridge and observed. After incubation, the Casein milk agar plate is observed for the zone of hydrolysis, which is a clearing around the bacteria streak and stabs. When looking at the plate the results show that there was hydrolysis so the test is positive.

The Nitrate Reductase test tube labeled #64B was removed from the fridge and the results were observed. The test tube showed there was growth so the next step was to add 8 drops of Nitrate A, the results did not produce a change so 8 drops of Nitrate B was added to the test tube. The results were observed and the solution in the test tube turned red, the test produced a positive outcome meaning that the organism being evaluated does have the ability to reduce nitrate. The growth streak plate labeled #68 was removed from the fridge and observed. Based on the observation the unknown bacteria did grow while in 45°C conditions. With this test result the first unknown microorganism was identified as Enterococcus faecalis. Based on the results of the Casein and Nitrate Reductase tests the two unknown organisms required a maltose test. Two test tubes containing the maltose solution were obtained and a small sample of #64A was taken from the nutrient streak plate and placed in one of the maltose test tubes and labeled #64A and placed into the 30°C inoculator for 36 hours and then placed into the fridge. The same procedure was executed for sample #64B, both test steps were followed according to the laboratory manual (Leboffe and Pierce).

November 27th, 2023

Both maltose test tubes were removed from the fridge and the results were observed. The testing tube labeled #64A turned yellow showing it had an acidic reaction. With an acidic reaction, another test was needed

A starch nutrients plate was obtained and a small sample of unknown bacteria was taken from the nutrient streak plate labeled #64B a single line was drawn and then another sample was collected from the streak plate and stabbed three times into the starch plate, the plate was labeled and placed into the 30°C inoculator for 24 hours. Due to the limited time that was remaining for the unknown’s identification process a DNas test was also set up on the same day to identify the unknown bacteria within the proper time frame. A DNas plate was obtained and a small sample was collected from the streak plate labeled #64B, two lines were drawn onto the DNas plate, then it was placed into the 30°C inoculator for 24 hours.

December 1

st

The starch plate and DNas plate were pulled from the 30°C inoculator and the results were observed. The a- amylase test is used to identify bacteria that can hydrolyze starch using the enzyme a-amylase, a clearing halo around the growth will indicate a positive result. After observation of the starch plate after adding iodine to the plate, there was no halo surrounding the growth, showing a negative result. This led to the observation of the DNas plate which was pulled from the 30°C inoculator. After observation, there appeared to have growth on the plate which led to the identification of the third and final unknown bacteria. According to the flow chart, the final unknown microbe is Staphylococcus aureus.

Results: Table for unknown microorganism #64A Test Purpose Observation Results Gram Staining To determine the Gram reaction of the bacterium. The bacteria appeared to be dark purple/blue. Gram Positive Streak Plate To isolate colonies Produced small cream, pale color growth.

N/A

Catalase To determine the ability to degrade hydrogen peroxide by producing the enzyme catalase. When the hydrogen peroxide was added the bacteria produced bubbles. Positive Glucose Tests for acid and/or gas produced from carbohydrate fermentation. The color changed from red to yellow and it had bubbles. Acidic Sucrose To see if the microbe can ferment the carbohydrate sucrose as a carbon source. The color changed to yellow. (This is where I went wrong on my flow chart) Positive Casein It determines if the microbe can produce caseinase. There is a clearing surrounding the microbe. Positive Maltose Determine if the microbe can ferment the carbohydrate (sugar) maltose as a carbon source. The color turned red showing there was a reaction. Acidic Lactose To determine the ability of a microbe to ferment a specific carbohydrate. The color did not change. Negative

Table for unknown microorganism # Test Purpose Observation Results Gram Staining To determine the Gram reaction of the bacterium. The bacteria appeared to be dark purple/blue. Gram Positive Streak Plate To isolate colonies Produced small cream color colonies

N/A

Catalase To determine the ability to degrade hydrogen peroxide by producing the enzyme catalase. When the hydrogen peroxide was added the bacteria produced no bubbles. Negative Mannitol To see if the microbe can ferment the carbohydrate (sugar) mannitol as a carbon source. The color changed from red to yellow and had no bubbles. Acidic Other test (Growth at 45°C) To see if the microbe is a thermophile. Which means it grows best in higher temperatures. Growth occurred Positive

Flow chart for identifying unknown Microorganisms:

identifying the unknown microbe labeled #64B is a DNas test, which resulted in a positive result indicating the final unknown bacteria is Staphylococcus aureus. This completed the biochemical testing for all three unknown microorganisms.

Research of three unknowns

Enterococcus faecalis : Commensal Turned Opportunist Enterococcus faecalis, a gram-positive bacterium, is a member of the human gastrointestinal microbiota. Typically, a commensal organism, it plays a vital role in maintaining microbial balance in the gut. However, E. faecalis can transform under certain conditions into an opportunistic pathogen, causing infections, particularly in healthcare settings. The importance of E. faecalis lies in its resilience and adaptability. It thrives in diverse environments, including the harsh conditions of the gastrointestinal tract. As a commensal, E. faecalis contributes to the maturation of undigested carbohydrates, creating advantageous short-chain fatty acids (Massoli et al., 2021). However, when presented to other body destinations or when the host's resistant framework is compromised, E. faecalis can cause contaminations, including urinary tract diseases, endocarditis, and intra-abdominal diseases. Research on E. faecalis focuses on understanding the factors that trigger its transition from commensal to pathogen. Biofilm formation, antibiotic resistance mechanisms, and virulence traits are critical areas of investigation (Al-Dobardani et al., 2023). The bacterium's ability to form biofilms on medical devices contributes to its role in healthcare-associated infections. As a primary cause of nosocomial infections, E. faecalis emphasizes the importance of infection prevention strategies in healthcare settings. We are studying the genetic determinants of virulence and antibiotic resistance to aids in developing targeted therapies and infection control measures. Staphylococcus aureus : Commensal and Pathogen in Human Health Staphylococcus aureus, a gram-positive bacterium, is a versatile microorganism with a dual role in human health. It serves as a commensal, commonly colonizing the skin and mucous membranes, contributing to the complex ecosystem of the human microbiota.

S. aureus, a versatile bacterium, can transition from a benign commensal to a formidable pathogen, causing a spectrum of infections, from mild skin conditions to severe, life-threatening diseases. Its importance in human health is underscored by its adaptability and the intricate factors influencing this transformation (Hassan et al., 12). Diseases may manifest as skin and soft tissue diseases, abscesses, pneumonia, or rise to life-threatening sepsis. The acquisition of anti-microbial resistance, eminently in the frame of methicillin-resistant S. aureus (MRSA), presents an imposing challenge in clinical settings. Current investigations on S. aureus dig into comprehending its virulence factors, mechanisms of anti-microbial resistance, and the complex flow of intuition with the host immune framework. This multifaceted investigation is critical for advancing our understanding of S. aureus infections and creating successful restorative strategies. (Du, Xin, et al 759). Virulence factors such as toxins, adhesins, and immune evasion strategies contribute to its pathogenicity. The quest for effective treatments involves deciphering the genetic basis of antibiotic resistance and exploring alternative therapeutic options. As a significant contributor to healthcare-associated infections, S. aureus underscores the importance of infection prevention and control measures. Vaccines targeting specific virulence factors are being explored to mitigate the impact of S. aureus infections. Understanding the dynamic interplay between S. aureus and the host is crucial for developing targeted interventions and effective antibiotic stewardship. Bacillus thuringiensis : A Biopesticide Revolution Bacillus thuringiensis , a gram-positive, spore-forming bacterium, stands as a groundbreaking figure in the annals of agricultural pest control. Its revolutionary impact traces back to its discovery in 1901 by Japanese biologist Shigetane Ishiwatari. However, in the mid-20th century, B. thuringiensis gained prominence for its extraordinary ability to produce crystal proteins with inherent toxicity against a diverse spectrum of insect larvae. This bacterium's notoriety in agriculture lies in its unique biological weaponry. During sporulation, B. thuringiensis synthesizes crystal proteins, specifically delta-endotoxins, which function as potent insecticides (Ishiwatari 26). These toxins, upon ingestion by susceptible insects, lead to the formation of pores in their gut lining, resulting in cell lysis and eventual mortality. This targeted and environmentally friendly approach has catapulted B. thuringiensis into a leading role in integrated pest management, offering an effective alternative to

Work Cited

Leboffe, Michael J and Burton E Pierce. Microbiology Laboratory Theory & Application Brief (3rd edition). Morton Publishing Company, 2016. Ishiwatari, S. (2019). A Remarkable Bacterial Disease of Silkworms. Journal of the College of Agriculture, Imperial University of Tokyo, 7(1), 19-27. Jallouli, Wafa, et al. "Review on biopesticide production by Bacillus thuringiensis subsp. kurstaki since 1990: Focus on bioprocess parameters." Process Biochemistry 98 (2020): 224-232. Du, Xin, et al. "Staphylococcus epidermidis clones express Staphylococcus aureus-type wall teichoic acid to shift from a commensal to pathogen lifestyle." Nature Microbiology 6.6 (2021): 757–768. Hassan, A. B., Tanko O. Hussaina, and Yemisi Adegboye. "Investigation of Staphylococcus Species from Anterior Nares of Healthy Individuals in Federal Medical Center Gusau." Gombe Technical Education Journal 13.1 (2021). Al-Dobardani, Qasim, Amera M. Al-Rawi, and Khairul A. Radzun. "Enterococcus faecalis a Major Cause of STD: A New Evidence." Rafidain Journal of Science 32.3 (2023): 1-8. Massoli, M. C. B., Cardozo, M. V., Ferroni, L. B., Casagrande, M. F., Nascimento, G. M., Pollo, A. S., & Iturrino, R. P. S. (2021). Enterococcus spp. Survival Through the Use of Standard Protocol for Clostridium Sp. Isolation. Brazilian Journal of Poultry Science , 23.