MEDIA PREPARATION AND CULTURE TECHNIQUE

 INTRODUCTION 

    

    Growth media or culture media was prepared to support microorganism growth such as bacteria, fungi (yeast and molds). It was a specific formulation and mixture of nutrients and other substances which were also being utilized for quality control test or determination of effectiveness by antimicrobial agents. The ingredients of culture media according to tests being performed and also the microorganism interest. Basically, the culture media can be in liquid (nutrient broth), and mixed with agar to be prepared in a petri dish either in the form of semi-solid or solid.  

    In addition, the preparation needs to undergo carefully and precisely. The main function is to ensure that microbial growth is promoted. It was important to follow the manual for the instructions as a requirement to fulfill the accuracy. In our lab practice, there were 2 types of media that are usually being used and prepared by students. There are PDA (Potato Dextrose Agar) and NA (Nutrient Agar). PDA is specifically being used to isolate fungi while the NA targeting bacterial isolation.

ACTIVITY 1: Media preparation for Potato dextrose Agar (PDA) and Nutrient Agar (NA)

MATERIALS:

2 Conical flask (250 ml)
Measuring cylinder
Spatula/spoon
2 sterilized petri dish
PDA powder

PROCEDURE:

1. Prepared 200 ml of PDA and also 200 ml of NA.

500 g PDA = 12.8 l (12800 ml

            x  g = 200 ml 

            12800 (x) =  (200 x 500)
            x         = 100000 / 12800
                                        = 7.8 g

            23 g NA = 1 l 
            x  g         = 200 ml (0.2 l)
            1 (x) =  (0.2 x 23)
            x        = 4.6 / 1
                                        = 4.6 g

2. Weight is ready to use PDA powder parallel to the volume of water.
3. Measured 200 ml distilled water, poured it into a conical flask with medium, and shook well to dilute the PDA powder.
4. Sterilized the prepared solution in an autoclave at 15 p.s.i., 1210c for 15 – 20 min. The medium needs to have been sterilized to avoid contamination by eliminating microorganisms.
5. After sterilization, let the medium cool down. Poured the medium into Petri dishes in the laminar hood.

    The prepared media was collected and brought to the autoclave station for further process. In addition, we were also introduced to the ready culture media (liquid) consisting of slope culture, radakan culture, and broth culture.

   

Figure 1: Example of radakan culture in a test tube

Figure 2: The actual shape of slope culture

Figure 4: Example Durham tube was used in nutrient broth culture

ACTIVITY 2: Culture technique (streak plate technique)

    The streak plate technique was used to isolate the mixture of bacteria into a pure culture. The group of bacteria was reduced as it was streaked over the agar surface. As a result, the individual bacteria separate from each other.

  Furthermore, the purpose of the streak plate technique is to produce divided bacteria. Due to morphology study and differentiation properties between each organism.

Figure 5: The sequence of a streak plate technique

  

PROCEDURE:

1. Sterilize the inoculating loop in the bunsen burner by putting the loop into the flame until it is red hot. Allow it to cool.

2. Pick an isolated colony from the agar plate culture and spread it over the first quadrant (No. 1)

3. Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just streaked, extend the streaks into the second quarter of the plate.

4 Then proceed to streak numbers 3 and 4.

5. Incubated the streak plate at 37°C for 24 hours. The colony was examined and recorded. If there are more colonies formed, streak again until the pure culture is obtained.

ACTIVITY 3: Media treatment for both PDA and NA (control, open, touch, cough and water)

PROCEDURE:

1.5 sets of petri dishes were prepared. while 1 set consists of 2 petri dishes of NA and the other 2 for PDA.
2. The treatments were for control, open, touch, cough and water test.
3. For control, both the agar need to be sealed after labelling and try not to open the lid.
4. Open test agar was done by getting the petri dish opened in the air in 2 minutes. Then, it can be sealed and labeled. 
5. Touch the agar with fingers (depending on our hygiene and it was free)
6. Coughed into the agar in one set of plates then closed back.
7. Distribute the distilled water gently in the agar using an inoculation loop and hockey stick
8. Sealed all the agar plates and label them according to the groups.

Figure 6: Our group members were sealing the plates using parafilm.

9. Store the plates with all the treatments in 3-7 being stored at room temperature.

Figure 7: One of our members arranged the plates for storage.

RESULTS:

	PDA	NA
Control
Open
Touch
Cough
Water

DISCUSSION

All the objectives of the activities were achieved to introduce and get to know the microbial species which can be cultured in the laboratory. The first activity helped the student to undergo the real process in making the medium agar from dehydrated media. Significantly, there are many types of dehydrated media and it is important to check the correct requirement before starting the process. The calculation needs to be standardized with the label from the manufacturer. 

PDA is specifically being used to isolate fungi. Nutrient agar or NA was applicable to isolate certain types of bacteria. It contains the essential nutrient to supply the variety growth of bacteria. The other function of NA to test antibiotic sensitivity as it produces the bacteria needed.

Furthermore, liquid culture also has its own purposes. In radakan culture (Figure 1), there is a tube or bottle containing solid media. There has been inoculated with a straight needle into it. Next, for the slope culture (Figure 2) the organism was cultured in solid media which had been coagulated in slope. The purpose of the test, to diagnose and show the growth pattern by lateral organisms. Meanwhile, organisms in broth culture (Figure 3) were grown in liquid media and sometimes used Durham tubes to detect production gas.

Moreover, the aseptic technique significantly contributes to successful results in microbiology tests. Foreign microorganisms or unwanted microbes could interfere with the process and delay the outcome. From activity 3, we can conclude that there are many microorganisms in our surroundings either bacteria or fungi, which can cause a threat to the process. An example of an aseptic technique, make sure the tools or other apparatus were clean, sterilized, and carry out the experiment in laminar airflow.