In the first session, you attempted separating S. aureus and E.coli bacteria from a mixed culture. Examine your plate after incubation at 37C. for the production of clumps of growth (colony forming units, c.f.u.s , 'colonies' or 'clones') on the plates. Is there evidence of two different bacterial species on the plate? Observe colony morphology and colour for each of the two bacteria present in the mixed culture and complete the table below:
TABLE I. Separation of S. aureus & E. coli from a mixed culture
Round, white, large colonies, smooth and raised
Punctifom, yellow, small colonies, smooth and raised
What is a bacterial colony and why it is referred to as a colony-forming unit (C.F.U.)?
A bacterial colony is a visible gathering of organisms growing on the surface of or within a solid medium. It is referred to a CFU, as it is comprised of over a million individual bacteria, all descendants from a single bacterium.
B.1 SELECTIVE MEDIA
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In the previous session, you have plated Staphylococcus aureus and Alcaligenes faecalis on both nutrient agar plates and on plates containing nutrient agar supplemented with 3M NaCl.
Observe and contrast the characteristics of any growth on the plates in the table below:
TABLE II. Growth selection of S. aureus and A. faecalis by NaCl
NA NA + NaCl
Notes: S.aureus, yellow, Punctifom and smooth
A.faecalis white/colourless, Punctifom.
B.2 DIFFERENTIAL MEDIA
You inoculated E. coli and P. vulgaris on McConkey agar. Compare the growth characteristics of both species and record your observations in the table below:
TABLE III. Differentiation of E. coli and P. vulgaris on McConkey agar
Notes: McConkey has a pH indicator which changes colour with pH.
E.coli use lactose which forms lactic acid and changes the pH, the colonies were round, large, smooth and raised
P.vulgaris produces NH4 and changes the pH in the McComkey agar and the indicator changes colour.
How does the McConkey medium work to differentiate between lactose fermenting bacteria and those that do not?
This medium is an indicator medium and a low selective medium. It contains bile salts and crystal violet dye, which hinder gram-positive bacteria, neutral red dye which stains microbes fermenting lactose, lactose and peptone.
Lactose fermenting bacteria such as E.coli produce red colonies. E.coli used lactose and forms lactic acid as a byproduct, as the lactic acid has a low pH this lowers the pH of the agar medium and changes colour due to the indicator present.
Non-lactose fermenting colonies for instance salmonella utilise peptone, this forms ammonia and increases the pH of the agar therefore leads to the formation of white/colourless colonies.
Contrast selective and differential media.
Selective media are intended to cultivate specific bacteria but not others. It selects for a certain feature allowing bacteria to grow for example, Mannitol Salt Agar (MSA) selects for the growth of halotolerant bacteria. The medium has a high salinity. Therefore only bacteria that can grow in a high level of salinity can grow and stay alive, for example S.aureus.
Differential media is designed to demonstrate the difference between different organisms grown on it for example on McConkeys agar some organisms look pink while others look colourless
C GRAM STAINING
Pure plate cultures of S. aureus and E. coli were provided to your group to stain using the Gram staining technique that should classify these bacteria as Gram positive or Gram negative.
Observe your Gram stained bacterial smears in the microscope using the immersion oil lens taking great care not to damage the microscope. Read the Appendix and ask advice from your Tutor on how to use the oil immersion lens. After use, carefully wipe oil immersion lens objective with lint free paper.
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Record bacteria colour (pink-red or purple violet) and microscopic shape (coccus or bacilli) for each of your Gram stained smears
TABLE IV. Separation of S. aureus & E. coli from a mixed culture
Bacteria colour Microscopic shape
What is the cellular basis for the Gram staining technique? Illustrate your answer with a diagram.
The bacteria that is gram positive has a cell wall that is dense 20-80 nm thick, this is 60-90% peptidoglycan. It has frequent interconnecting layers of peptidoglycan. Also in the cell wall are teichoic acids, which go throughout the cell wall and are comprised of phosphates, sugar alcohol ribitol and polymers of glycerol. A few have a lipid attached. The external surface of the peptidoglycan has proteins, which vary with species of the bacteria. Whereas gram negative cell walls only contain 10-20% peptidoglycan and is surrounded by a outer membrane,
In gram-positive bacteria the crystal violet and iodine combine to form a large molecule that precipitates away from the cell. The alcohol mixture causes dehydration of the peptidoglycan consequently decreasing the space between the cell wall to entrap the crystal violet complex inside the cell. Whereas in gram-negative bacteria the alcohol mixture dissolves the lipid outer membrane of the cell wall and damages the membrane on which the peptidoglycan in attached and the cell is decolourised.
D. MICROSCOPIC OBSERVATIONS
You used and/or observed a number of stained bacterial smears during Practical 1 and were advised to find out about the taxonomy, biology and importance of each of the microorganisms observed.
Use the Table below to record the most important features of bacteria species observed.
TABLE V. Importance of bacteria species observed using compound microscope
Bacillus, gram -ve
Synthesizes vitamin K for the liver
Cocci, gram +ve
Found in nose and on skin, capable of producing staphyloxanthin
Bacillus, gram -ve strand
Converts most pathogenic forms of arsenic and arsenite to a less dangerous form, it is found in soil and water, also in respiratory tracts of people with cystic fibrosis.
Bacillus, gram -ve,
Ferment sugars in anaerobic conditions. Degrades urea to ammonia. Inhabits the intestinal tracts of humans and animals.
Cocci, gram -ve, flagellated. Pathogen of Typhoid fever. Found in humans chickens and cattle
Produces antibacterial substances,
Fix nitrogen in root nodules of legumes. Found in soil.
Bacillus, gram -ve, compact
Found in soil, found in chains where the cells are joined together by polysaccharides on the cell walls. Can survive extreme weather conditions. Produces penicillin amidase, which is needed for making penicillin. It produces enzymes for modifying corticosteroids.
Bacillus, green stranded
Cynobacteria. Found in salted waters, photosynthetic.