Introduction to Microbiology (University College London)
Laboratory report: experiment 1 Culture medium preparation
Liquid media used for culturing microorganisms in the lab is typically sterilized using a small autoclave (essentially, a programmable pressure cooker).
I prepare 5 litres of medium, split this into five one-litre bottles and put these into the autoclave. The autoclave is programmed for a 20 minute/121oC cycle.
My lazy colleague also makes 5 litres of the same medium, but instead puts the whole lot into a single large bottle and autoclaves this using the same programme.
A day-or-so after autoclaving, the medium in my colleague’s bottle has gone cloudy indicating that contaminating bacteria have survived the autoclaving and have grown in the medium. In contrast, the medium in all five of my bottles is clear (i.e. no bacterial growth).
The 5-litre bottle is so large that heat transfer to the interior of the solution in retarded. As a result, the entire solution is not heated up to 121oC for sufficient time. By contrast, if splitting them into 1-litre bottles, the total surface area is larger such that the contents of the bottles are sufficiently heated to the required temperature within the 20-minute cycle.
I would either programme the cycle of autoclave to be longer or set it to a higher temperature. This either allows more time for heating up the bottle, or the bottle can be heated up faster due to larger temperature difference.
Laboratory report: experiment 2 Reducing bacterial contamination
|Results Report your findings. What did the bacterial growth look like on the control (water only) plate? Which of the various treatments were effective in reducing the amount of bacteria? Was there a difference between the Gram positive M. luteus and the Gram negative E. coli? The size of clear zones (diameter in mm) of different treatments were measured as follows:|
|11 mm||6 mm||12 mm||6 mm||none||6 mm||none||none|
|The data of another group using M. luteus are as follows:|
|22 mm||9 mm||20 mm||none||none||10 mm||none||none|
|The class results are as follows (% of groups showing inhibition):|
|Combining the three sets of results above – bleach, TCP, isopropanol are the most effective means of inhibiting bacterial growth (most groups have inhibition). They are all commercial products for disinfections. Soap & vinegar also show good inhibition. Salt solution & honey show some extent of inhibition, but are probably not effective means of bacterial control. All groups had bacterial growth on the control plate; the whole surface of the control plate appeared to be cloudy. Comparing the two sets of data using E. coli & M. luteus, bleach & isopropanol are the most effective treatment for inhibiting growth of two species. TCP & vinegar also inhibit growth of these two species. Soap shows inhibitory effect on E. coli but not on M. luteus, although this maybe due to experimental variations only, so whether the treatments have differences on the two bacteria is inconclusive.|
Colony growth and analysis of two bacterial strains
Laboratory report: experiment 4 Isolation of Pseudomonas fluorescens
By visiting this webpage, answer the following questions about Pseudomonas fluorescens.
a) Which explosive pollutant of soil could P. fluorescens degrade? Draw the structure of this compound.
P. flurorescens degrades TNT (trinitrotoluene) explosive. Its systematic name is 2-methyl-1,3,5-trinitrobenzene, and its structure is as follows:-
b) Explain the two ways that P. fluorescens might act to suppress plant diseases.
P. flurorescens produces some secondary metabolites, including antibiotics, siderophores and cyanide, which protect them from fungal infection. P. flurorescens also colonises rapidly in soil, outcompeting other pathogenic species.
c) How big (in Mb) is the genome of P. fluorescens strain PfO-1 (hint: click on ‘Info’)?
d) If a typical protein-coding gene is ~1 kb, approximately how many genes would you expect P. fluorescens to have?
Approximately 6438 genes.
e) Hospital infections of Pseudomonas species occur frequently and can occasional prove lethal (see: http://www.bbc.co.uk/news/uk-northern-ireland-16779722). Approximately how many cases of infection are reported by the Health Protection Agency each year?
Around 3850 cases (between 3700 and 4000) are reported each year.
Laboratory report: experiment 6 Isolation of Bacillus from soil
Using ‘Todar’s Online Textbook of Bacteriology’ (http://www.textbookofbacteriology.net/)
Go to the contents page and then click on ‘Bacillus and related endospore-forming bacteria’ at the bottom of the page. Then answer the following:
a) Explain why Gram staining of spore-forming species might prove problematic.
Endospores formed are strongly resistant to staining. Hence, endospores may appear as non-staining entities. This may affect the identification of properties of species.
b) Mature endospores have no detectable metabolism. What is this state called?
This state is known as crytobiotic.
c) List five differences between vegetative cells and endospores.
1) Vegetative cells appear to be refractile under microscope while endospores appear to be non-refractile.
2) Vegetative cells have high cytoplasmic water activity while that in endospores are low.
3) There are enzymatic activities and macromolecular synthesis in vegetative cells while they are absent in endospores.
4) Vegetative cells are generally sensitive to lysozyme actions, dyes and staining while endospores are resistant.
5) Endospores have high resistance towards heat, acids and radiation while vegetative cells are susceptible to attack.
Report your findings. What was the appearance of the colonies on the plate? What percentage were lactose fermenters? What was your estimate for the number per ml of river water? How did this compare with the group results?
The number of colonies on the plate with undiluted river water were all clustered together and too many to be counted. Colonies in the plate after the first dilution were still too many to be counted. There were 28 distinct colonies on the plate with a 100- fold dilution; there were only 2 distinct colonies on the plate with a 1000-fold dilution.
Thus we consider the plate with 1000-fold dilution. The distribution of morphologies of the colonies is shown below.
|Number of colonies||Percentage|
Lactose fermenters gave red/pink colour due to production of acid, while those bacteria that cannot ferment lactose produces ammonia using peptone, giving a white colour. For our sample, 64% of bacteria were lactose fermenting while the remaining 36% were not.
The estimated number of Enterobacteria in the river water sample is 28×103 = 28,000 per mL. Compared to the class results, which is 85,000 per mL, our results were lower. This may probably due to error in carrying out serial dilution.
Laboratory report: experiment 8
Skin bacteria and their antibiotic sensitivity
Report your findings: the number and types of bacterial colonies observed before/after handwashing, etc.; the results of the analysis of the chosen colonies – Gram stain, morphology, etc. To which antibiotics were the bacteria resistant/sensitive?
The bacteria colonies found on the first sector that was pressed with unwashed finger were abundant. They were mostly white in colour; their sizes vary from <0.5 mm to 2 mm. There were only a few bacteria colonies found on the second sector that was pressed with washed finger. They were white in colour and their sizes were all less than 0.5 mm. On the third sector that was taken from the surface of a mobile phone, bacterial colonies were abundant. They were mostly red in colour, only one colony was white in colour. The sizes for the red colonies were <0.5 mm while for the white colony was 2 mm. Similar results were obtained from sector 4.
On viewing under microscope, the white colonies on sector 1 and 2 were spherical in shape and Gram-positive. The red colonies on sector 3 and 4 were rod-shaped and Gram-negative.
A white colony was chosen for the antibiotic test. The sizes of the clear zones are as follows.
|Antibiotic used||Quantity used||Size of clear zone|
|Tetracycline||10 µg||14 mm|
|Penicillin||1.5 units||8 mm|
|Streptomycin||25 µg||No clear zone|
|Rifampicin||5 µg||10 mm|
|Vancomycin||30 µg||10 mm|
Therefore for the chosen colony, it was sensitive to all chosen antibiotics except for streptomycin.
Antibiotics work by inhibiting a specific metabolic process found in bacteria cells.
Describe the mode of action of the following two antibiotics and briefly explain why human cells are unaffected.
Penicillin is a type of β-lactam antibiotics, which inhibits cell wall synthesis. During cell wall synthesis, an enzyme called transpeptidase cross-links peptide chains. In the presence of penicillin, transpeptidase bind to penicillin and cannot continue to catalyse the cross-link reactions. As cell wall synthesis still continues, newly synthesised cell wall is not cross-linked and the strength is weakened. In addition, the binding of enzyme to penicillin also stimulates enzymes known as autolysins, which digest existing cell wall. Bacterial cells are eventually self- degraded. Human cells do not have cell walls, so are unaffected by penicillin.
Streptomycin is a type of aminoglycosides, which is a natural antibiotic produced by a bacteria called Streptomyces griseus. It inhibits protein synthesis, it binds to the 30S subunit of bacterial ribosome and interfere with tRNA binding to the 30Ssubunti. This results in misreading of codons, eventually inhibits protein synthesis and causes bacterial cell death. The structure of ribosomes in human cells (eukaryotes) are different from that of bacterial cells (prokaryotes), therefore human cells are unaffected by action of streptomycin.