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Microbiology Lab Report

NAME: MYTHILY STUDENT NO: A0075269U

Introduction
According to the British Pharmacopeia - Preparation for oral administration: a) The total viable aerobic count for bacteria should not be more than 10^3 and the viable count of fungi should not be more than 10^2 per gram or millilitre of oral preparation. b) There should be no presence of E Coli in the preparation at all.

Objectives of Experiments:
To determine the total viable microbial count in the pour plate (bacterial and fungal media) is within prescribed limit of BP (<1000cells/ml) for Oral Preparation Rhinathiol Promethazine 125mL. To detect presence of E.Coli in for Oral Preparation Rhinathiol Promethazine 125mL. To study Gram staining Method to differentiate Gram Positive and Gram Negative bacteria under light microscope.

Materials & Methodology:


a) Instruments/Apparatus 1. Petri dishes (20 pcs) 2. 3 Universal bottles labeled as A, B & C. 3. Bunsen Burner 4. Sterile Glass Spreader 5. Disposable Spreader 6. Pipette 7. Wire Loop 8. Microscope 9. Slide 10. Colony Counter b) 1. 2. 3. 4. 5. Other materials Iodine Alcohol Dilute Carbol-fuchsin Diluents (Sodium chloride-peptone solution of pH 7) Product X : Rhinathiol Promethazine 125mL

6. Mac Conkey Agar plate 7. Casein Soya bean digest Agar (CSA) 8. Sabouraud-glucose agar.

Figure 1 : Mac Conkey Agar

c) Methodology

Preparation of dilutions:
Prepare 3 bottles as following: Bottle A contains 10 mL of product X + 90 mL of diluents ( 1:10) Bottle B contains 10 mL from bottle A + 90 mL of diluents ( 1:100) Bottle C contains 10 mL from bottle B + 90 mL of diluents ( 1:1000)

Prepare 9 petri dishes for bacteria count experiments.

Remove the cap of the bottle of melted nutrient agar Casein Soya bean digest Agar. Pour the contents into the 9 Petri dish and replace the lid.

Lift the lid of the Petri dish and place 1mL of the dilution from bottles A,B and C in the centre of the dish and replace the lid. Repeat 3 times for each Bottle A,B and C.

Mix the contents of the Petri dish with a sterile glass spreader, moving in a figure of eight, but avoiding any splashing over the edge. Let the agar set to form a pour plate.

Repeat the step 1 to 3 for fungal count studies. But replace CSA with Sabourand glucose Agar.

Place the CSA containing petri dishes in the incubation room. Meanwhile, the SGA agar plates in the room temperature.

After 4 days, check the CFU for all the petri dishes by using colony counter

Preparation of Smear
Wash slide with alcohol/acetone

Heat the loop for 3 secs.Place a small loopful of water on the centre of the slide

Heat the loop for 3 secs. Place a small portion of the culture to the drop of water.

Mix the sample using the loop,spread thinly over the slide. Hold the slide with forceps and pass,film upper most twice through the Bunsen flame to fix.

Staining Method
Prepare a mixed of E-coli and S.aureus

Stain with Gram's stain for 1 min. Remove excess stain,wash with iodine and leave it for 1 min. Repeat this step twice.

Wash with alcohol until decolourised. Then, rinse with water. Counter stain with dilute carbol-fuchsin for 30 secs. Wash with water, blot, dry and examine.

Microscopic Examination
Fix the slide to the microscope stage Place a drop of immersion oil on the selected area of the smear, open the iris diaphragm fully and rack the condenser right up. Swing the oil-immersion objective into position.

Set your eyes at stage level, rack down using coarse adjustments until objective touches the oil.

Adjust brightness. Focus sharply with the fine adjustments.

Adjust the iris diaphragm and rack down the condenser slightly, if necessary to give even illumination.

Methods to detect presence of E.Coli in oral preparation- Rhinathiol Promethazine 125mL The presence of E.Coli was tested by culturing the oral prepartion on Mac Conkey agar medium and left in the incubator for 4 days. The MacConkey Agar is as selective medium, using the bile salts and crystal violet dye to inhibit Gram-positive bacteria while at the same time enabling Gram-negative bacteria to grow well. Thus, aany growth of red, non mucoid colonies of gram negative rods would indicate the possibilities of E. Coli presence. Upon which, need to be further clarified and verified by biochemical test or staining method.

Results & Discussions


a) Microscopic Examination of Microbs Gram stain is used routinely as a differential stain so that you can classify bacteria as "gram positive" or "gram negative". Gram positive bacteria stain a blue purple colour because they retain the stain-iodine complex inside their cells. Gram negative bacteria stain a red colour because they have cell walls which allow the stain-iodine complex to be washed out of the cell by alcohol.

Thus, based on our observation of the cells under the microscope, we noticed that E. Coli appears to be cocci or rod shaped and red in colour. Thus, proving it is gram negative bacteria. Meanwhile we are able to also observe more circular-shaped Gram positive bacteria, S.aureus that appears deep purple or blue under the light microscope.

Figure: Photos showing the Gram positive ( blue or purple coloured ones), S.aureus and Gram negative ( red coloured ones ), E-coli under miscrope.

b) Determine the viable counts of unspecified organism in oral preparationRhinathiol Promethazine 125mL.

Bottle No A1 A2 A3 B1 B2 B3 C1 C2 C3

No of Dilution Factor Colony Total No of colonies 1/10 5 1/10 3 (5+3+1)/3*10 = 30 1/10 1 1/100 0 1/100 2 (0+2+0)/3*100 = 67 1/100 0 1/1000 1 1/1000 2 (1+2+0)/3*1000= 1000 1/1000 0 Table 1 : Agar Medium : CSA for total viable bacterial count

Based on the results, we can deduct that viable aerobic count is 30 cfu/mL in Bottle A, 67 cfu/mL in Bottle B and 1000 cfu/mL in Bottle C. Thus, bacteria concentration in all 3 dilutions are within the prescribed limit in BP ( 1000 cfu/mL). However, the results in Bottle B and C indicates that there have been sampling error as Bottle A should contain the highest number of viable bacteria count since it is the most concentrated solution.

Dilution No of Bottle No Factor Colony Total No of colonies A1 1/10 0 A2 1/10 0 undetected A3 1/10 0 B1 1/100 0 B2 1/100 0 undetected B3 1/100 0 C1 1/1000 0 C2 1/1000 0 undetected C3 1/1000 0 Table 2 : Agar Medium : SGA for total viable fungal count Meanwhile, findings for fungal presence in oral preparation shows that no viable fungal count.

c) To detect presence of E.Coli in oral preparation- Rhinathiol Promethazine 125mL.

No growth was detected in the Mac Conkey agar which means E. Coli is not present in the oral preparation. Even though no growth was detected in Mac Conkey agar, the results for E.Coli could have been more reliable if we have conducted the microb revival and enrichment step.

Conclusion
The oral preparation, - Rhinathiol Promethazine 125mL has passed the 2 important tests: a) total viable aerobic count which was below prescribed limit, 1000cfu/ml b) Absence of E. Coli This concludes that it is considered safe to be consumed.

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