Microbiology

We can classify bacteria by theur shape and by the Gram staining technique.

There are 4 shapes of bacteria:

• Cocci/spherical e.g. Staphylococcus
• Bacillus/rod-shaped e.g. Escherichia
• Spiral/corkscrew-shaped e.g. Spirillum
• Vibrio/comma shaped e.g. cholera

Furter diffentrition is possible by the way the bacteria tend to be grouped e.g. single, in apirs, clusters, or forming chains.

Gram staining: A method of staining bacteria as an aid to their identification.

This divides bacteria into gram-positive and gram-negative and this difference is due to chemcial compositon of their cell walls.

• Crystal Violet- stains all bacteria violet
• Lugols Iodine- fixes stain more permanently into the cell wall.
• Acetone/alcohol- washes off violet dye from gram-negatve, leaving only gram positive coloured.
• Sufranin- counterstain: stains gram-negative bacteria red, but positive remains violet.

Gram positive bacteria:

More susceptible to anibiotics, penicillin and lysozymes than gram-negative bacteria. Examples includm Bacillus, Staphylococcus and Streptococcus. Pencillin works by interfering with the links of peptidogylcan, causing the cell wall to be weak and collapse during cell division. 

Gram negative bacteria:

Have more complex cell walls where the peptidoglycan cell wal is supplemented with lipopolysaccharide layers. This does not retian dyes like cyrstal violet. Example is salmonella. They are resiatnt to lysozyme and penicillin. Antibiotics against them work by interfering with protein making mechanisms (which are different to human cell mecahnisms.) 

Microrganisms can divide very quickly if they need to (bacteria cn divide every 20minutes if in the right conditions.)

Conditions:

• Temperature- growth is regulated by enzymes which genrally work best within a range of 25-45 centigrade. Mammalian pathogens work best at 37 degrees.
• pH- bacteria prefer slightly alkaline conditions, and fungi acidic.
• nutrients: e.g. agar, which includes carbon (glucose), nitorgen (organic and non) vitamins and mineral salts. Nitorge is needed to produce amino acids in protein synthesis.
• oxygen- many micororganisms need oxygen to grow. Clostridium is an anaerobe and couses moist gangrene in wounds.

Aseptic: a procedure performed under sterile conditions.

To prevent contamination of the cultures:

• Sterilise all equipment, apparatus and media before use.
•  Handle cultures carefully and use specific equipment (sterile loops) to prvent contamination.

To prevent contamination of the environment:

• Sterilise work surface before and after experiment suing Lysol 3%$ or another disinfectant.
• Use correct handling techniques to protect perosnnel and environment.

• Grasp culture bottle in one hand and remove lid using little finger of other hand, and do not place it down.
• Flame mouth of bottle for 2-3 seconds.
• Pass loop through flame until red hot.
• Lift the lid of petri dish just enough to allow loop through.
• secure petri dish with tape , but not all way round as oxugen needs to get in-- (prevents pathogenic/diseas causing microorganisms growing)
• incubate around 25 centigrade (not 37 centigarde as this is prime temp fo pathogens)
• Do not open petri dishes after incubation.
• Equipment is then heated in an autoclave at 121centigrade for 15 minutes. This is necessary as some pathogenic bacetria can create an internal resting cell called a spore, whoch contains DNA, and is resitsant to heating, dessication and pH changes. They can later reform into bacterial cells.
• Disposable equipment should be autoclaved then binned.
• Gamma radiation is another form of sterilisation

Directly:

Rough estimates can be made by measuring the diameter of a colony at regular intervals as its spreads.

Plating and Counting:-- finds VIABLE CELL COUNT

The cells in a very small sample are counted and this result is then multiplied to give the popukation desnity per cm3 of culture. Even then, cell counts can be too high, so the solution is dilute 10 fold-- serial dilution.

A water authority my use this to estimate the extent of contamination. They would use serial dilution and then add 1cm3 to an agar plate. This is then incubated for 2 days at 25centigarde.  The colonies are then counted and multiplied up by the dilution factor.

If the dilution was insufficent, the colonies will merge, called 'clumping'. Counting will be an underestimation. 

This method relies on the assumption than each colony arised from the asexual reproduction of a single cell.

Haemocytometer:--- TOTAL cell count

This uses a specialist microscope slide, but cannot define between dead and alive cells.

Turbidimetry:--TOTL cell count

A colorimeter measures the cloudiness or tubidity of the culture as cell numbers increase. This is then comapred to a standard graph of light absorbance.

Fermentation is widely used to create large amounts of bacteria or fungal cells e.g. for the proudction of penicillin and other antibodies.

Advantages of growing microorganisms in fermenters:

•  Rapid growth without supplied enzymes
• lower temperatures
• cheaper

Main principles

• A pure culture of the organism for formation and harvesting of a pure product.
• Suitable conditions for efficient growth and efficiency.
• Sterilised vessel, including all entrances and outputs. Sterile medium used. Aseptic technique is necessary to maintain purity.
• Force aeration to increase growth of aerobes, and could also mix the culture to increase contact with nutrients. A sparger can be used by an air inlet.
• Temperature and pH monitors used to maintain ideal conditions. Water cooling jacket used to remove excess heat producd by the bacteria.

• The fermenter is inoculated with penicillum notatum, which then grows in optimum conditions.
• It takes about 30 hours for competion to start and penicillin to be produced. It si secreted by the fungus and accumulates in the medium. It takes a while because penicillin is a secondary metabolite.
• Afyer about 6 day the fluid is filtered , pencillin is extracted and purified. The culture medium is maitained and processed.

A disadvantage of thsi method is that the fermenter has to be emptied and sterilied before a second batch.

Primary Metabolite: A direct product from the matabolising of glucose during all periods of the microbe life cycle. Continuous culture can be used to get these metabolites. Production can be maintained as products are constantly removed and nutriets are fed in. E.G. ALCOHOL AND INSULIN.

Secondary metabolite:  tehse are produced when food sources are becoming scarce. Pencillin notaum produces the antibiotic to remove competing bacteria.