Growth of Microorganisms

Experiment 1: Fluid Thioglycollate Medium to Assess the Effect of Oxygen on Bacterial Growth

Introduction

Microbes have different requirements for their growth. Fluid thioglycollate medium (FTM) is an enriched differential medium that is used to differentiate oxygen requirement levels of different organisms. Basically, the media has different oxygen levels which decrease with the increase in distance from its surface (Chauhan & Jindal, 2020). This is brought about by the reaction of sodium thioglycolate. The medium contains resazurin indicator which turns pink when oxygen is absorbed into the media and colorless in the absence of oxygen. The FTM medium is used to determine bacteria's growth characteristics based on their oxygen requirements.

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Objectives

To determine the oxygen growth requirements of bacterial samples obtained from various surfaces based on their growth location in the FTM tubes.

Material and Methods

Materials

The materials required included; 5 FTM tubes nutrient agar, microwave, bleach and permanent markers.

Lab wares used; 5 sterile cotton swabs, 5 cm petri dishes, test tube rack, parafilm, 5 sterile inoculation needles, a pair of disposable gloves, hot pad and scissors.

Methods

First, the nutrient agar plates were prepared. This was achieved by heating the nutrient agar bottle in the microwave and swirling it every 10 seconds to distribute heat and mix the liquefied mixture well. Its contents were then poured onto petri dishes, covered and left to solidify.

The second step was to inoculate the nutrient agar plates after they had solidified. Microbial samples for inoculation were obtained by swabbing the skin, nose, throat and shoe using sterile cotton swabs. The samples were inoculated by carefully streaking them onto the surface of the agar then sealed with a strip of parafilm. Each of the plates was labeled using a permanent marker as per the source of the microbial sample. The control plate was streaked using a sterile swab and labeled as control. The plates were then incubated upside down until there was sufficient growth of colonies to be inoculated on FTM tubes.

The third step was the inoculation of FTM tubes. Each of the tubes was labeled to correspond with the labeling on the nutrient agar plate. The tubes were then uncapped, placed on boiling water bath for 10 minutes then recapped. They were then left to cool before being inoculated by picking up an individual colony from the culture plate and using a sterile inoculating needle, inserting the needle straight to the bottom of the tube, mixing it with the medium by moving it up and down through the initial needle track then finally replacing the lid of the tube and sealing it with parafilm. The same technique was used for all the plates with their respective tubes while a sterile inoculation needle was used for the control tube.

Last, the tubes were inoculated in a warm place for three days then examined for microbial growth. The culture plates were stored for future use whereas the FTM tubes and control plates were disinfected using bleach before being disposed.

Results

Discussion and Conclusion

References

Chauhan, A., & Jindal, T. (2020). Microbiological Culture Media: Types, Role and Composition. In Microbiological Methods for Environment, Food and Pharmaceutical Analysis (pp. 23-66). Springer, Cham.

Lagier, J. C., Edouard, S., Pagnier, I., Mediannikov, O., Drancourt, M., & Raoult, D. (2015).

Current and past strategies for bacterial culture in clinical microbiology. Clinical microbiology reviews, 28(1), 208-236.

Experiment 2: Effect of Chemical Germicides on Bacterial Growth

Introduction

Chemical agents can be used to suppress or stop the growth of microorganisms. They are effective since microorganisms are sensitive to certain chemicals as they either block their metabolism or prevent the synthesis of essential micro molecules (Presterl et al., 2019).

Germicides are able to kill microorganisms upon contact but cannot completely eliminate them. Germicides that can be applied topically on biological tissues are referred to as antiseptics whereas disinfectants are germicides used on inanimate objects (Lachapelle, 2020).

Objectives

To determines the growth inhibition properties of chemicals.

Materials and methods

Materials

Materials used included 5 growth plates from experiment 1, nutrient agar, 4 filter paper disks, 10 ml hibiclens antimicrobial antiseptic hand cleanser, candle, matches, 6 ml sterile phosphate buffered saline (PBS), 10 ml distilled water, 70% isopropyl alcohol and bleach. Lab wares required included five 5 cm petri dishes, disposable gloves, forceps, 5 sterile transfer pipettes, 5 sterile snap-cap tubes, reusable metal inoculation loop, 5 sterile plate spreaders, (4) 15 ml screw-top tubes, test tube rack, ruler, parafilm, 10 ml graduated cylinder, 100 ml beaker, lab notebook and scissors.

Methods

Five agar plates were prepared and marked as skin lawn, nose lawn, throat lawn, shoe lawn and control lawn. The plates were then divided into four equal quadrants and each was marked as bleach, 70% isopropyl alcohol, Hibiclens and control.Five snap-cap tubes were labeled as skin, nose, throat, shoe and control, and approximately 1 ml of PBS poured into each of the tubes. The inoculation loop was sterilized by dipping it in isopropyl alcohol for 10 seconds then passing it several times through the flames of a candle.

The sterile loop was used to transfer a colony from the plate labeled as skin to the tube labeled 'skin' and mixed gently with the PBS. 0.1 ml of the solution was pipetted into the skin lawn plate and a sterile spreader used to distribute the solution evenly on the surface of the agar. The same procedure was used for the subsequent plates using their respective solutions as labeled. 0.1ml of PBS was used on the control plate. The new plates were set aside and the tubes disinfected using bleach before disposal.

10 ml of 10% bleach solution, 70% isopropyl alcohol, hibiclens and distilled water were transferred into a 15ml screw top and labeled as bleach, 70% isopropyl alcohol, hibiclens and control respectively. 16 pieces of filter paper were cut using scissors in the size 2.5 mm x 2.5 mm.

Forceps were sterilized by dipping it in isopropyl alcohol for 10 seconds then passing it several times through the flames of a candle. The sterile forceps were used to pick a piece of filter paper, dipping it in the 10 ml bleach solution, dripping off excess bleach from the filter paper, and placing it on each plate's bleach quadrant. The same procedure was used for the subsequent treatments.

The plates were then incubated in a warm place and observed for zones of inhibition after 3 days. The zones of inhibition were measured in millimeters and recorded.

Results

Discussion and Conclusion