Gram Positive and Gram Negative Bacteria Experiment

The aim of this experiment was to differentiate between Gram negative and Gram positive bacteria by Gram staining method. In addition, this experiment allowed us to visualise the morphology and arrangement of the bacterial cell.

Approach (to achieve aim):

In this experiment, two different staining methods were employed, the monochrome staining method and the Gram staining method. The monochrome staining method is a direct stain that stains the bacteria using only one type of stain. Gram staining method is useful for classification and identification of bacteria. The Gram stain is a differential stain which allows one to classify bacteria as Gram positive or Gram negative.

Materials and Methods:

Refer to page 4 and 5 for staining procedures.

Results:

Table 1 shows the monochrome and Gram Staining of Staphylococcus epidermidis and Escherichia coli

Test

Monochrome Staining

Gram Staining

Bacteria Cells

Staphylococcus epidermidis

Escherichia coli

Staphylococcus epidermidis

Colour

Blue

Blue

Purple

Shape

Cocci

Bacilli

Cocci

Arrangement

Grape-like clusters

Isolated

Grape-like clusters

Results

Both cells were stained blue due to the primary staining caused by Loeffler’s methylene blue

Gram positive bacteria

Conclusion and Discussion:

In the monochrome staining of S. epidermidis and E. coli, both the bacteria cells were stained blue in colour. This was due to the presence of rich nucleic acid and the negatively charged surface receptor molecules of the bacterial cell which attracted the cationic stain of methylene blue. Therefore, the cells retained the colour of the methylene blue stain and appeared blue.

In the Gram staining of S. epidermidis and E. coli cells, S. epidermidis appeared to be in purple colour. This indicates that S. epidermidis was a Gram positive bacterium. The cell wall of Gram positive bacterium had a thick peptidoglycan layer which contained magnesium ribonucleate and lacked lipid in the membrane system. During Gram staining process, the crystal violet basic stain formed crystal violet-iodine-magnesium ribonucleate complex on the Gram positive bacterium cell wall. Therefore, Gram positive bacteria appeared violet in colour due to the formation of the complex. In the case of E. coli, it was a Gram negative bacterium because it was stained red. The application of 95% ethanol dissolved the lipids in cell walls of Gram negative bacteria, therefore resulting in the outflow of crystal violet from the cell. In addition, the application of carbol fushin stain allowed E. coli to uptake the secondary stain and thus appearing red during observation.

Under the microscope, S. epidermidis was cocci shaped and had grape-like clustered arrangement. On the other side, E. coli was rod shaped and had isolated arrangement.

END

Name: Wei Xiao Yang

Question (i.e. Aim):

The aim of this experiment was to observe bacterial colony characteristics and the mixed bacterial culture background in nutrient agar plates.

Approach (to achieve aim):

In this experiment, streak plate method was used on nutrient agar plates to produce isolated colonies of bacteria from mixed culture.

Materials and Methods:

Refer to page 7-9 in the practical manual.

Results:

Table 2 shows the colony characteristics of A and B in solid medium

Mixed Culture

Colony Characteristics

A

Shape

Circular

Size

1-2mm

Chromogenesis

Colourless

Opacity

Translucent

Elevation

Raised

Surface

Dull

Edge

Entire

Texture

Smooth

Emulsifiability

Turbid

Gram nature

Gram negative

Odor

Negative

Consistency

Butyrous

Morphology

Bacilli

Arrangement

Isolated

Conclusion and Discussion:

From the experimental results, bacterial colonies from A were rod shaped, gram negative and had isolated type of arrangement when viewed under the microscope. Viewing the colony characteristics, the colonies from A appeared circular, colourless, translucent, raised, smooth, and had a larger size colony. In addition, the surface of colony A was dull and it had entire edges. Therefore, judging from the colony characteristics, morphology and Gram nature of the bacteria, bacteria from colony A could be possibly be E. coli. On the other hand, bacteria colonies from B were Gram-positive, cocci shaped and had grape-like clusters of arrangement. The colonies from B were circular, whitish, opaque, convex, granular and punctiform in size. Thus, bacteria colonies from B were most likely S.epidermidis.

END

Name: Wei Xiao Yang

Question (i.e. Aim):

The aim of this experiment was to observe the nature of growth of different bacteria in liquid medium.

Approach (to achieve aim):

In this experiment, identification of bacteria was based on the physiological properties of the bacterial growth in liquid medium.

Materials and Methods:

Refer to page 10 of the practical manual.

Results:

Microorganism

• Escherichia coli

Growth characteristics

Amount of growth

• Moderate

Surface growth

• Absent

Turbidity

• Present (uniform)

Sediment

• Present (disintegrate upon shaking)

Chromogenesis

• Negative

Gram nature

• Gram negative

Morphology

• Bacilli

Arrangement

• Isolated

Table 3 shows the growth characteristics of Escherichia coli and Pseudomonas aeruginosa in liquid medium.

Conclusion and Discussion:

From the experimental results, E. coli being a facultative anaerobic bacterium was able to grow in moderate amounts as it shows uniform turbidity in the liquid medium. The E. coli cells sediments at the bottom of the test tube due to the huge amount accumulation of dead cells. On the other hand, P.aeruginosa being an aerobic bacterium formed pellicle at the liquid-air surface and was turbid towards the surface. This would suggest that P.aeruginosa favoured aerobic respiration for growth as the liquid-air surface had higher amount of oxygen content as compared to the bottom of the tube. In addition to that, P. aeruginosa was able to produce pyocyanin pigments which would diffuse into the medium, turning the liquid medium bluish green in colour. Under the microscope, both E.coli and P.aeruginosa were Gram-negative; rod shaped and had isloated arrangement.

End

Name: Wei Xiao Yang

Experiment: Biochemical Test

Question (i.e. Aim)

The aim of this catalase test experiment was to differentiate Staphylococci bacteria from Streptococci bacteria due to the presence of catalase enzyme. The aim of this oxidase test was to detect the presence of cytochrome oxidase enzyme in the microorganism.

Approach (to achieve aim):

In the catalase test experiment, detection of catalase producing bacteria was done by observing the decomposition of hydrogen peroxide into water and oxygen. In the oxidase test experiment, the detection of cytochrome oxidase producing bacteria was done by redox reaction of cytochrome oxidase which turned the oxidase test reagent into purple colour.

Materials and Methods:

Refer to page 11-13 of the practical manual

Results:

Table 4 shows the catalase Test results of Staphylococcus aureus and Streptococcus faecalis

Test

Catalase Test

Bacterial Cells

Staphylococcus aureus

Streptococcus faecalis

Observation

Effervescence formed

No Effervescence formed

Results

Positive catalase test

Negative catalase test

Table 5 shows the oxidase Test results of P. aeruginosa and E. coli

Test

Oxidase Test

Bacterial Cells

Pseudomonas aeruginosa

Escherichia coli

Observation

Development of dark purple colour at the site of smear

No colour change is observed

Results

Positive oxidase test

Negative oxidase test

Conclusion and Discussion:

In the experiment of catalase test, S. aureus being catalase positive demonstrated the ability to catalyse the degradation of hydrogen peroxide to oxygen and water, hence producing effervescence. On the other side, S. faecalis being in the streptococci family does not possess the catalase enzyme to degrade the hydrogen peroxide. Therefore, there was no effervescence produced. The catalase test allows us to differentiate between staphylococci species from streptococci species.

In the experiment of oxidase test, P. aeruginosa being oxidase positive possess cytochrome c oxidase which oxidised N, N, N′, N′HYPERLINK “http://en.wikipedia.org/wiki/Wurster’s_blue”-tetramethyl-HYPERLINK “http://en.wikipedia.org/wiki/Wurster’s_blue”pHYPERLINK “http://en.wikipedia.org/wiki/Wurster’s_blue”-phenylenediamine (an artificial electron donor for cytochrome c) in the oxidase test reagent. The presence of cytochrome oxidase catalyses the transport of electrons from NADH to electron acceptors (oxygen) and reduced it to water. Therefore, the oxidation of N, N, N′, N′HYPERLINK “http://en.wikipedia.org/wiki/Wurster’s_blue”-tetramethyl-HYPERLINK “http://en.wikipedia.org/wiki/Wurster’s_blue”pHYPERLINK “http://en.wikipedia.org/wiki/Wurster’s_blue”-HYPERLINK “http://en.wikipedia.org/wiki/Wurster’s_blue”phenylenediamine formed a dark, oxidized product called indophenol blue, turning the oxidase test reagent soaked paper strip into maroon colour. On the other hand, E. coli showed no colour change in the paper strip. Hence, this suggested that E. coli did not have any cytochrome oxidase activity and was oxidase negative.