Macroscopic and Microscopic Characterization
Colony Morphology Identification
Colony morphology identification is a macroscopic characterization observed visually. Observation of colony morphology from isolates was carried out by identifying colonies based on their morphology. Isolates on SCA agar medium were initiated by opening the petri dish to determine different colony types. The observed colony morphology included substrate mycelium, aerial mycelium, pigment, and elevation. The morphology identification process was done by visual observation, and once completed, the petri dish was sealed with plastic wrap and placed back into the incubator.
Gram Staining
The Gram staining procedure was performed to determine bacterial cell shape using a microscope, including coccus, bacillus, and spiral. Another purpose of Gram staining is to determine whether the observed bacteria are Gram-negative or Gram-positive. The working steps of the Gram staining method are as follows: prepare the required tools and materials, then perform fixation by sterilizing the object glass with distilled water and a Bunsen burner flame. Bacterial isolate was taken using a sterile inoculation loop and smeared on the object glass. The bacterial smear was then dropped with reagent A (Crystal violet) and left for 60 seconds, then rinsed with distilled water and dried. The next step was dropping reagent B (Iodine) and leaving it for 60 seconds, then rinsing again with distilled water and drying. The decolorization process was carried out by dropping reagent C (decolorizer), then leaving it for 60 seconds and rinsing with distilled water before drying again. The final step was dropping reagent D (safranin) and leaving it for 60 seconds, then rinsing with distilled water and drying. The dried sample on the object glass was then observed under a microscope with 1000x magnification to determine the color and shape of bacterial cells.
Biochemical Test
Biochemical tests performed for Actinomycetes bacterial characterization used the catalase test and oxidase test.
a. Catalase Test
The catalase test was performed by sterilizing the tools and materials to be used and the UV LAF for 30 minutes. The object glass was sterilized using a sterilizer and labeled with the sample code at the end of the object glass. The next step was sterilizing the inoculation loop using an infrared sterilizer and letting it cool. The Actinomycetes colony to be tested was taken using an inoculation loop and smeared on the object glass, then H₂O₂ was dropped onto the colony smear. The result of the drop was observed to see whether air bubbles appeared or not. A positive catalase test result is indicated by the formation of air bubbles, and a negative catalase test result is indicated by the absence of air bubbles. All tested isolates were documented to determine which isolates were negative and positive.
b. Oxidase Test
The oxidase test was performed to determine whether Actinomycetes bacteria can produce oxidase enzymes or not. The oxidase test process began by sterilizing the tools to be used and the UV LAF for 30 minutes. In this research, the oxidase test did not use an Oxidase Test Strip, but instead used 3% KOH solution. The glass slide was labeled according to the Actinomycetes bacterial isolate code. The next step was sterilizing the glass slide near a Bunsen burner flame to avoid contamination. Then one isolate colony was taken using a sterile inoculation loop and smeared on the glass slide. The smeared bacteria were then dropped with 3% KOH and the inoculation loop was moved slowly over the smear. The bacterial smear was left for 5 seconds and the color change was observed. A positive oxidase test result is indicated by a change in the drop producing slime, and a negative oxidase test result is indicated by no slime found in the drop. The final step was documenting the oxidase test results for all isolates.
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