Differential and Biochemical Tests to Identify Pathogenic Microorganisms

1 Differential and Biochemical Tests INTRODUCTION Pathogenic microorganisms are the primary causes of diseases. These microorganisms are found on almost all surfaces. They not only inhabit but also thrive in all environments on earth.

Microorganisms have a unique metabolic capacity that enables them to utilize a wide range of substances as their sources of energy (Dhanasekaran & Jiang, 2016). Human beings and other living organisms cannot metabolize most of the substances utilized by microorganisms. Studies have shown that some microorganisms live in organism substances whereas other thrives in inorganic substances (Murray, Rosenthal & Pfaller, 2020).

Microbiologists focus on metabolic capabilities and biochemical reactions to determine the type of microorganisms thriving on various surfaces (Kumavath, 2017). Whereas some biochemical tests are run independently following the preparation of sample culture, others are integrated into the differential growth media. Differential and biochemical tests allow for the examination of various microorganisms’ properties such as nutrient requirement, metabolism, susceptibility to antibacterial agents and presence of hydrolytic enzymes.

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2 OBJECTIVES The objectives of these exercises were to determine the presence of microorganisms containing cytochrome oxidase on various surfaces. The exercise was also intended to identify the presence of aerobic microorganisms on different surfaces. The third objective was to determine the sensitivity of microbes on different surfaces to antibiotics.

METHODS AND MATERIALS: Experiment 1: Oxidase Assay For Respiration The materials utilized in this experiment were nutrient agar, microwave, distilled water, permanent marker, bleach, isopropyl alcohol, scissors, lab notebook, Parafilm, inoculation loop, Petri dishes, sterile cotton swabs, and candle. The initial procedure in this experiment was the preparation of the agar media. The agar was then put into two Petri dishes and allowed to condense for 30 minutes (Esciencelabs LLC, 2019). A permanent marker was used to draw a line at the back of the Petri dishes. The four halves were then labeled as shoe, sink, control or phone.

A sterile cotton swab was then moistened and swabbed in the bathroom sink. The cotton swab was gently streaked on the surface of the agar on the half labeled as “sink” (Esciencelabs LLC, 2019). This procedure was repeated with the shoe and phone swabs. Finally, a sterile cotton swab was moistened and gently rubbed on the control part of the Petri dishes. The plates were then sealed tightly using a Parafilm and incubated upside down for 3 days. The inoculation loop was sterilized using isopropyl alcohol and candle flame. The loop was used to transfer the colony on the sink, shoe and phone parts of the dishes to the DrySlide assay (Esciencelabs LLC, 2019). The assay was closely observed and the color change was recorded in the table below.

RESULTS The results indicate that various surfaces including the sink, shoes, and phone contain microorganisms with cytochrome oxidase.

3 Table 1: Assay Results Colony sample

Color change

Oxidase outcome

Sink

Yellow to purple

Positive

Shoe

Orange to yellow

Negative

phone

Yellow to purple

Positive

Control

No change of color

N/A

METHODS AND MATERIALS: Experiment 2: Catalase Assay The materials used in this exercise were distilled water, permanent marker, isopropyl alcohol, microwave, 12 drops Hydrogen Peroxide, and nutrient agar. The labware included wax pencil, Parafilm, Petri dishes, inoculation loop, pipette, hot pad, scissors and lab notebook. The agar medium was prepared as the first step of the exercise. 5 ml of the agar was then put in two Petri dishes and allowed to condense. The bottom of the Petri dishes was separated into two halves using a permanent marker (Esciencelabs LLC, 2019). The 4 halves were labeled as shoe, sink, phone and control. Moistened cotton was used to swab the sink, the phone and the shoe surfaces.

The cotton swab was then gentry streaked on the surface of the agar according to the source of the swab. A sterile cotton swab has then rubbed the surface labeled as the control on the Petri dishes. A Parafilm seal was then placed on the Petri dishes. The plates were incubated at room temperature for three days. A third petri dish without agar was taken and divided into three sections labeled shoe, sink and phone (Esciencelabs LLC, 2019). Two circles were drawn on the labeled sections using a wax pencil. An inoculation loop sterilized with isopropyl alcohol was used to scoop colonies from the agar plates and transfer them to the circles on the labeled

4 sections of the third plater without agar (Esciencelabs LLC, 2019). Two drops of hydrogen peroxide were poured on the circles for all samples and the observations recorded on the results table.

RESULTS Table 2: Assay Results Petri dish section

Colony

Bubbles

Catalase

Sink

1

Yes

Positive

2

Yes

Positive

1

Yes

Positive

2

Yes

Positive

1

Yes

Positive

2

Yes

Negative

Shoe

Phone

METHODS AND MATERIALS: Experiment 3: Antibiotic Sensitivity Materials used in this exercise were nutrient agar, yeast, sugar, distilled water, microwave, matches, warm water, permanent marker, distilled water, bleach, candle, and sterile saline. Labware comprised Parafilm, forceps, Petri dishes, rule, test tubes, disposable gloves, sterile cotton swabs, measuring spoons, scissors, hot pad, Mueller Hinton agar and 3 Tetracycline Disks. An inoculation loop, test tube rack, pipettes, 250 mL beaker and a lab notebook were also used (Esciencelabs LLC, 2019).

The procedure for this exercise included the preparation of the agar media and the agar plates. The beaker was then used to measure 230mL of water. A packet of yeast together with 1 tablespoon of sugar was then poured into the beaker and mixed gently. The permanent marker was used to separate the Petri dishes into three sections labeled as Skin,” “Local,” and “Yeast.”

5 sterile cotton was moistened and rubbed on the skin surface of the forearm. This swab was streaked softly on the agar surface labeled as skin (Esciencelabs LLC, 2019). A similar procedure was followed to obtain a sample from the doorknob. The doorknob swab was gently streaked on the surface of the agar labeled local. 10 drops of yeast were poured onto the palm of a gloved hand. A cotton swan was rubbed on the palm covered with yeast and then streaked on the agar plate section labeled yeast (Esciencelabs LLC, 2019). The closed agar plates were sealed with Parafilm and incubated for three days at room temperature.

The next procedure was the preparation of the Mueller-Hinton Agar Plates. Three standing test tubes were labeled as skin, yeast and local and 2 mL sterile saline poured into each test tube. A sterile inoculation loop was used to transfer colonies into respective test tubes. A sterile cotton swab was dipped into the test tubes and then rubbed on the respective outer sections of the Mueller-Hinton agar plates. Antibiotic discs, labeled as Ampicillin-AM10, Tetracycline-Te30 and Kanamycin-K30, were placed on to the samples using forceps. The plates were incubated for three days at 37.7 °C (Esciencelabs LLC, 2019). The final exercise was the measurement of the zones of inhibition using a ruler and results were recorded in the table below.

RESULTS Table 3: Skin Sample, Zone of Inhibition Antibiotic disc

The diameter of the zone (mm)

AM10

21

K30

23

Te30

29

Table 4: Zone of Inhibition for Local Sample

6 Antibiotic disc

The diameter of the zone (mm)

AM10

0

K30

17

Te30

26

Table 5: Zone of Inhibition for Yeast Sample Antibiotic disc

The diameter of the zone (mm)

AM10

20

K30

21

Te30

27

DISCUSSION The exercises were effective in determining the presence of microorganisms on different surfaces. In the first exercise, the chromatic reducing agent which is a component of the oxidase reagent changed color due to oxidation (Murray, Rosenthal & Pfaller, 2020). This property is used to test the presence of microorganisms containing the cytochrome oxidase enzyme. The test is read quickly as the reagent might turn purple upon exposure to the surface. The catalase test, on the other hand, uses hydrogen peroxide to test the presence of aerobic microorganisms. The exposure of a colony to a high concentration of H2O2 leads to the formation of gases (Murray, Rosenthal & Pfaller, 2020). The gas bubbles are, thus, used to indicate the presence of catalase in the colony. Sensitivity to antibiotics also helped to determine the presence of microbes on surfaces. The zone of inhibition was different for different antibiotics as well as the sample of microorganisms (Kumavath, 2017). The size of the zone of inhibition indicates the sensitivity of

7 the microorganisms to the agent use. Whereas a small zone indicates a low sensitivity a larger zone indicates a higher sensitivity.

8 References Dhanasekaran, D., & Jiang, Y. (Eds.). (2016). Actinobacteria: Basics and Biotechnological Applications. BoD–Books on Demand.

Esciencelabs LLC. (2019). Differential & Biochemical Tests. https://www.esciencelabs.com/sites/default/files/V3/Microbiology/2nd_Edition_Microbio logy/MB_6200_L09_DifferentialBiochemicalTests/story_html5.html Kumavath, R., (2017). Antibacterial Agents. BoD – Books on Demand.

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2020). Medical Microbiology E-Book. Elsevier Health Sciences.