FUAD HASAN

BTEC National Certificate

Section 1

The microorganism term is used instead of microbes; they are very small organisms sizeand not be seen by naked eye, belonging to various groups: Bacteria, Fungi, Protozoa, one can add Viruses, characterized by their noncellular structure.

The micro-organisms are probably the first alive beings appeared on ground, they would be thus the common ancestors of all the living being. Although microorganisms appeared 3.8 billion years ago, they are observed by Pasteur and koch through their microscope way after their apparition. Pasteur delivered the fatal blow to the doctrine of spontaneous generation thanks to his examination and Koch discovered revealed the existence of bacilla at the origin of tuberculosis. Those discoveries changed radically the perception of the human beings on the world.

I as technician have been given a task by my head techncian, the task is to to isolate and analyse microbial samples from immediate environment and I should include:

  • Analysing effectiveness of commercially disinfectants and antiseptics on an individual bacterial species.
  • Morphological properties
  • Examining the bacterial colonies on nutrient agar plates as well as their cellular characteristics (microscopically) by the use of gram stain.

Section 1: Isolation of microorganisms from the environment

Materials

  • Petri dish containing a layer of sterile nutrient agar.
  • Inoculating loop
  • Bunsen burner
  • Marker pen
  • Sealing tape
  • Soil sample
  • Sink basin (tank water)
  • Air in the lab
  • Glass rod

Procedure

  1. Prepare the three Petri dishes and agar (under aseptic conditions) for inoculation. With a marker, label the bottom of the plates with the date, the name of the sample to be inoculated.
  1. Obtain a suspension of the soil sample by dissolving the soil in distilled water. Sterilise the inoculating loop. Dip the cooled sterilised loop into the soil suspension then use it to make zigzag streaks across the surface of the agar. Tilt the edge of the Petri dish to keep out unwanted bacteria and close the lid as quickly as possible to avoid contamination. Secure the lid with short piece of sealing tape. Do not seal all the edge.
  1. Immerse a swap in sterile water and swap the inside of a sink. Rub the swap over about 3-6 cm2 of the agar taking care not to introduce microorganisms from the air. Secure the lid with short piece of sealing tape.
  1. For sampling the air, remove the lid from a plate and allow to sit uncovered for about 45 minutes. Then close the lid. Secure the lid with short pieces of sealing tape.
  1. Incubate all plates inverted at 370 C. leave the plates until the next session.

Results

Sample name / Number of colony in each sample
Air / 21
Water / 86
Soil / 65

Conclusion of Isolation of microorganisms from the environment session

From this session, I can see microorganisms had grown in all three different environments. From the result above I see fish water had the highest amount of colonies, from my observation the fish water whilst I was doing this experiment I could see is quiet dirty.

Section 11: microscopic examination and separation of bacteria colonies.

Materials

  • Agar plates from section 1
  • Ruler

Procedure

  1. Examine your plates
  2. Count, if possible, or estimate the total number of organisms on each plate
  3. Determine the number of different organisms based on differences in colony type on plates (e.g. round, concentric, wrinkled, irregular etc)
  4. Describe the morphology of a few well-isolated colonies on each plate

Colour- yellow, white, colourless, opaque, etc

Size- measure diameter in mm

Shape- round, concentric, wrinkled, irregular etc

Texture- shiny, dull, mucoid, hairy, etc.

Margin or edge- entire, wavy, labate, irregular, filamentous

Laboratory report of microscopic examination and separation of bacteria colonies session.

1. What environment was sampled? Soil sample

Number of colonies 74Number of different colonies 5

Colony type I / Colony type II / Colony type III
Colour / White / White / Colourless
Size (diameter in mm) / 3 / 1 / 5
Shape / Round / Concentric / Round
Texture / Hairy / Dull / Dull
Margin or Edge / Wavy / Entire / Entire
Elevation / Umbonate / Flat / Raised

Soil sample

Colony type I: was White in colour, measured 3 mm in size, round shaped very hair Texture. The Margin was wavy, Elevation was Umbonate. Colony type I: was White in colour, measured 1 mm in size, concentric shaped Dull Texture. The Margin was Entire, Elevation was flat. Colony type III: was White in Colour, measured 5 mm in size, round shaped and Dull Texture. The Margin was Entire, Elevation was raised. All Colonies turned out to be different.

2.What environment was sampled? Tape water sample

Number of colonies 135 Number of different colonies 9

Colony type I / Colony type II / Colony type III
Colour / White / Yellow / cream
Size (diameter in mm) / 2 / 5 / 6
Shape / Round / Round / Concentric
Texture / Shiny / Dull / Shiny
Margin or Edge / Entire / Entire / Entire
Elevation / Flat / Flat / Flat

Water sample

Colony type I: was White in colour, measured 2 mm in size, round shaped very Shiny Texture. The Margin was Entire, Elevation was flat. Colony type I: was Yellow in colour, measured 5 mm in size, Round shaped Dull Texture. The Margin was Entire, Elevation was flat. Colony type III: was brown in Colour, measured 6 mm in size, Concentric shaped and Shiny Texture. The Margin was Entire, Elevation was flat. Unlike the soil sample which mostly all Colony turned out to be different, quiet few thing turned out the same such as the Margin or Edge and Elevation.

3. What environment was sampled? Air sample

Number of colonies 30 Number of different colonies 5

Colony type I / Colony type II / Colony type III
Colour / orange / yellow / white
Size (diameter in mm) / 6 / 8 / 5
Shape / Concentric / Round / Concentric
Texture / Shiny / Shiny / Dull
Margin or Edge / Entire / Entire / Entire
Elevation / Flat / Raised / Raised

Air sample

Colony type I: was Yellow in colour, measured 6 mm in size, Concentric shaped very Shiny Texture. The Margin was Entire, Elevation was Flat. Colony type I: was White in colour, measured 8 mm in size, Round shaped Shiny Texture. The Margin was Entire, Elevation was raised. Colony type III: was Cream in Colour, measured 5 mm in size, Concentric shaped and Dull Texture. The Margin was Entire, Elevation was flat. The only thing that was same all three colony was Margin or Edge which was all entire

Conclusion of examination and separation of bacteria colonies session
In session of the experiment I had to analyse my plate and the separation of bacteria colonies. Then record the outcome of the experiment of physical appearance (Colour, Size, Shape, Texture, Texture Margin or Edge and Elevation) of colonies. The outcome of this session of the experiment I think was successfully
Sample name / Number of colony in each sample
Water / 135
Air / 30
Soil / 74

The above table illustrates number of colonies produced by each sample; significantly the water produced a much higher number of colonies compared to sample of air and soil. Going back to when I was doing session of this experiment, I remembered that the fish tank water was quiet dark in colour compared to drinking water. So I went enquire with the laboratory technician in charge of changing fish tank water, and asked how often it was changed and she replied a average of 4 -5 days. So I came to a conclusion, the sample probably had been collected on late day of its recent changing of fresh water. So I think this factor alone played a role in why the fish tank water produce significantly much higher number of colonies compared to sample of air and soil

Section III: Microscopic examination and identification of isolated cells

Apparatus

  • Plates from session 1
  • Microscope slides
  • Sterile water
  • Inoculating loop
  • Bunsen burner
  • Gram staining reagents Crystals violets (primary stain)

Gram’s iodine

95% ethanol

Satranin (counter stain)

  • Microscope
  • Immersion oil

Method

  1. Prepare a heat-fixed smear of two different bacterium (one from each inoculated plate onto separate microscope slides
  2. Place a small drop of sterile water on each side
  3. Using an inoculating loop that has been sterilised over flame and cooled, pick a small sample of a bacterium from a single, well isolated colony.
  4. Suspend it in the drop of water and spread the bacterial suspension over the slide until the middle half of the slide is covers.
  5. Choose other types of single, well isolated sample colonies from the other plates (microbial samples) and repeat the procedure on other slides. Let the slides air-dry.
  6. after the have dried, using a holder, grasp one edge of the slide and pass the slide quickly through the bunsen flame three times to heat-fix the bacteria. Place the slide on a staining rack over the sink.

Gram stains the bacteria on the slides as follows:

  1. Flood the slide with the crystal stain and allow to incubate for 1 to 2 minutes.
  2. Wash stain off with a gentle stream of tap water.
  3. Remove excess water by shaking the slide.
  4. Flood the slide with Gram’s iodine stain and allow to incubate for 1 minute.
  5. Wash stain off and remove excess water as above.
  6. Tilt the slide and gently decolourise with several drops (not more than 4-5) of 95% ethanol), allowing it to flow over the slide and drop off one corner. Rinse off alcohol as before.
  7. Apply the safranin stain and allow to incubate for 2 to 3 minutes. Wash stain off as above. Gently blot the slide on a paper. Wipe the bottom of the slide to remove excess stain that may decrease visibility.
  8. Examine the microorganisms on the slides using oil-immersion lens of the microscope.
  9. Record your observations. Draw what you see under the microscope. Identify the microorganisms as either gram-positive or gram-negative bacteria.

Cellular Analysis by Gram Stain of An Isolated Microorganism

1. Microscope magnification used? Oil magnification

Gram positive

bacteria that is dark stained blue or violet by Gram staining, in comparison with Gram-Negative bacteria,which is not pobsible to retain the stain, which instead soak up the counterstain and gives a appearance of red or pink

Gram negative

Gram-negative bacteria is bacteria that don’t tain crystal violet dye in the Gram staining protocol Gram-positive bacteria which will retain the crystal violet dye when washed in a decolorizing solution.

Conclusion

To conclude this session, which I had to analyse and identify two samples of cells. Then sketch my observation in microscope and then attempt to identify whether it was a gram positive or negative bacteria. The outcome of this session, one was gram positive and gram-negative bacteria.

The diagrams of Microscopic examination and identification of isolated cells on the next page.

Section IV: Effect of disinfectants on microorganisms preparation

Apparatus

  • TSA plate containing environmental sample from section 1
  • Inoculating loop
  • Bunsen burner
  • Nutrient broth in culture tube.

Method

  1. Using an inoculating loop that been sterilized over the flame of a Bunsen burner and cooled, pick up a small sample of a bacterium from a single, well isolated colony from your plate containing the environment sample
  2. Suspend the colony in the nutrient broth (make sure the colony’s in the broth).
  3. Label the tube with your initial and the date and environment sampled. Place the tube in a rack and incubate at 370C 18-24 hrs.

Section V: effects of commercially available disinfectants and antiseptics on an isolated microorganism.

Apparatus

  • Inoculated nutrient broth from session 11
  • Large plate
  • Sterile cotton swabs
  • Antiseptics/disinfectants to be evaluated:
  • Bleach
  • Others
  • 95% ethanol
  • Scope
  • Rubbing alcohol
  • Antibacterial soap
  • Others of your choice: Dettol, Hand wash, Bleach, Washing up liquid
  • Sterile water
  • Forceps beakers
  • Filter-paper disks

Method

  1. Label the large plate with your initial and the date.
  2. Check your nutrient broth that you inoculated last week for bacterial growth.
  3. Carefully swirl the tube and if the broth is cloudy, your organism grew.
  4. Obtain a sterile cotton swab, making sure you do not contact the swab portion.
  5. Again carefully swirl the tubes containing your test organism to resuspend it in the broth.
  6. Wet the cotton swab with the test microorganisms and gently, so as not to mess up the agar, spread the organisms uniformly over the entire surface of the large plate.
  7. Select 4 antiseptics/disinfectants to be tested. If the material to be tested is a solid, such as toothpaste, prepare slurry in a tube using some of the agent and sterile distilled water.
  8. Since you will be testing 4 antiseptics on the plate, plan ahead the location of the disks to make sure they will be well distributed on the surface of the agar.
  9. Sterilize the forceps by dipping it into 95% alcohol and then passing through (do not hold over) the flame of a Bunsen burner to burn off the alcohol. Let the forceps cool
  10. Pick up a sterile filter-paper disk and dip it into the antiseptic solution to be tested. Remove the excess fluid from the disk by touching the disk to the side of beaker. Avoid placing an excess of liquid on the disks.
  11. Place the disk on the surface of the inoculated plate and gently pat the disk in place with the forceps.
  12. Label the bottom of the plate below the disk to identify the agent being tested.
  13. Repeat the procedure with the other antiseptics/disinfectants to be tested. Remember to mark below each disk the name of the agent being tested.
  14. Incubate the plates inverted at 370C for 18-24 hrs.

Session VI: analysis of commercially available disinfectants and antiseptics on an isolated microorganism.

Apparatus

  • Inoculated plates with antiseptic
  • Disinfectant disks from session III ruler.

Method

  1. Measure the diameter (mm) of the zone of inhibition of growth

2. Compare the effectiveness of each of the agents tested.

When interpreting the results, keep in mind that the agents tested may diffuse to varying degrees in the agar medium used. Therefore, this type of testing, while simple, only provides relative effectiveness data.

Questions and answers

  1. Did you have growth in your broth culture? Yes I did
  1. Effect of antiseptics tested

Antiseptics tested / Zone of inhibition (diameter in mm)
Bleach / 0. 9mm
Dettol / 1.3 mm
Hand wash / 0.5 mm
Washing up liquid / 1.0 mm
  1. Conclusion pf antiseptics tested on ability to ‘kill germs on contact’

In comparison the most effective antiseptics tested was the hand wash, because microorganisms grown around it, whilst the other antiseptics (dettol, bleach and washing up liquid) which where less effective.

  1. Do you think antiseptics tested are bactericidal or bacteriostatic?

The antiseptics which are bactericidal are Dettol, bleach washing up liquid and hands wash is bacteriostatic.

Analysis

From analysing my results above, my observation illustrate that hand wash had the smallest size in diameter, because the microorganisms grew around it. From the outcome of the experiment, I found dettol antiseptic which was able to kill bacteria very well. On the other hand, hand wash wasn’t very able to kill the bacteria as much. Bleach wasn’t able to kill the bacteria, because bleach purpose is to whiten or chemical remove, thought a process called oxidation.

Conclusion

In this last session, I measured from each microoganism of the four antiseptics I had used. The outcome of this are listed above in the table. From the four antiseptics, the bleach came out which largest diameter.

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