Objective: In these experiments, we aimed to perform fundamental microbiology laboratory techniques. These include; testing the effectiveness of face masks; understanding the effectiveness on handwashing and direct transmission, performing a gram stain; performing a streak plate for isolation, and reviewing colony morphology and cell descriptions.

Results:

Procedure (I): Effectiveness of Masks

In the observations of performing a cough without a barrier grew visual circular opaque.The TSA plate of a cough with barrier showed a singular circular colony.                                   

Figure 1. TSA plates demonstrating colonies formed after a 24-hour incubation at 37°C.

Procedure (II): Hand washing and Direct transmission through handshake

 After incubation, direct transmission from person to person showed dense opaque colonies. The TSA plate demonstrating colonies after handwashing have dispersed circular colonies that are opaque.

Figure 2. Direct transmission TSA plate (left), after hand washing (right) after 24 hours of incubation in 37°C

Procedure III: Gram Staining E.coli and S. epidermidis

 After oil immersion, culture showed both gram negative and positive strains of bacteria. Gram positive bacteria were consistent with purple stained dicocci cell morphology. Gram negative culture showed pink single bacillus cell morphology.

Figure 3. Gram stain under 100X oil immersion

Procedure IV: Streak plate for isolation

 In performing a streak plate isolation, isolated colonies were grown.

Figure 4. Streak plate after incubation for 24 hours at 37°C.

Discussion:

 Reviewing andPreforming fundamental microbiological techniques aided in making laboratory practice safe and efficient. On the TSA plate where no barrier was used to prevent microbes form colonizing, more culture was grown, on the media where a protective barrier was used, only a single colony was observed. While this colony can be accounted for from contamination from the experimental bench, testing the effectiveness of masks demonstrated that when there is a protective barrier like a mask to block bacteria between source and receiver less pathogens are transmitted. By the implementation of protective barriers in health care settings and/or school, we can expect to see less bacterial and viral infections through direct contact.

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 In the comparison of direct transmission to handwashing, the TSA plate of direct transmission showed dense white opaque colonies. These colonies decreased in density as we got to transmission to quadrant 4. In observing the colonies on the TSA plate after handwashing, colonies drastically decreased in density and appear more dispersed. This dispersion is to be expected, as implicated by research, washing hands with soap and water decreases microbes pathogenic and nonpathogenic on the area of contact (1).

After gram staining the culture of E.coli and S. epidermidis, we expect to see both gram positive and gram negative cultures on the slide. After 100x oil immersion, we were able to successfully stain S. epidermidis with crystal violet and use a mordant to increase the stains adhesion to the cell wall. To stain the gram negative culture of E.coli we used a secondary stain as after decolorating, the gram negative cells are not able to remain stained due to the thinness of the bacteria’s cell wall. As shown in figure 3, the pink bacteria (E. coli) show a colony margin that is single bacillus, which is consistent with expectations. The purple stained bacteria (S. epidermidis) has a colony margin that is dicocci. Our conclusions are supported by the Staphylococcus genus, as most of the species derived from this genus are consistent with cocci type morphology (2).

In performing individual steak plate to isolate independent colonies, there were several factors accounting for the inconsistency in gradient across the five sectors. The first is inaccurate quenching from sterilizing heat source to TSA media. There is likelihood that quenching was not sufficient and when streaking section to section killed the cultures from previous sections. Due to the variable transition section to section, the isolated colonies could be accounted for by contamination and improper aseptic technique.

In future experiments more technique review must be done to reinforce basic biochemical test and aseptic techniques. In obtaining more consistent practices, more reliable results are to be expected.

References

(1)   Burton, M., Cobb, E., Donachie, P., Judah, G., Curtis, V., & Schmidt, W. (2011). The Effect of Handwashing with Water or Soap on Bacterial Contamination of Hands. International Journal Of Environmental Research And Public Health, 8(1), 97-104. doi: 10.3390/ijerph8010097

(2)   Foster T. Staphylococcus. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 12. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8448/

References Link cited for Discussion questions

Discussion Extra credit Questions

Under the universal precautions mandate, what bodily fluids besides blood must be treated as positive for HIV and Hepatitis?

Under the Universal Precautions Mandate, all bodily fluids including; semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid saliva in dental procedures and any bodily fluid that is contaminated with blood must be treated as positive for HIV and Hepatitis as in certain situations it is impossible to differentiate between body fluids (1).

What are the main types of transmission of microorganisms? Give an example of each.

The main types of transmission of microorganisms are

Direct contact: involves transmission of microorganisms from person to person. Ex: an uninfected person coming into contact with an infected person (STI’s), infected person sneezing on uninfected person.

Indirect contact: involves transmission of microorganisms via vector, food and water, contaminated object (table).

Differentiate between vehicle vector and vertical transmission.

Vehicle transmission: transmission of pathogens via food, water, and air.

Poor water sanitation and dust can serve as vehicles of transmission

Vector transmission: transmission of pathogens via an agent that carries and transmits a pathogen.

Mosquitos that carry malaria.

Vertical transmission: transmission of pathogens from mother to baby.

Vertical transmission can occur through the placenta to the baby, breast milk, and direct contact at the time of birth.

A lab mate drops a test tube of V. cholera on the floor and it shatters. How would you handle the spill?

While wearing proper protective clothing, cover area with paper towels. From the outside and working my way in with a disinfectant completely cover the area and allow contact time according manufactures instructions. Dispose of all biohazard material in biohazard bins and containers, autoclave or appropriately disinfect equipment used to clean up spill.

How many different biosafety levels for microorganisms are there? Give one example of each.

There are 4 biosafety levels for microorganisms.

BSL-1: Lactobacillus acidophilus

BSL-2: Staphylococcus aureus

BSL-3: Lyssavirus

BSL-4: Zaire ebolavirus

Research and describe the cellular and colony morphology of two pathogenic bacteria

E. coli: gram-negative rod-shaped bacterium. Their colony morphology on nutrient agar tends to be thick, grey smooth opaque or translucent discs.