4.4 MICROBIOLOGY
1. Bacteria may be round (coccus), rod shaped (bacillus), spiral
shaped (spirillum). Shape has traditionally been used for classification
along with metabolic reactions.
2. The shape of bacteria is due to their rigid cell wall which has a
unique structure.
3. The Gram stain may be used to differentiate
between Gram positive and Gram negative bacteria
according to whether the cell wall retains crystal
violet (stains purple), or not (stains red by the
counterstain).
Gram staining is used to differentiate between Gram +ve
and Gram –ve Bacteria The different staining properties are
due to differences in the chemical composition of their cell
walls. First the sample is Fixed, the Crystal violet is added.
Both retain the Crystal Violet at this point in time. Then
Iodine is added and Ethanol. The ethanol breaks down the
Lipopolysaccharide layer in the Gram Negative bacteria, and
so they become decolourised. The counter stain – Safarin, is
then added and stains the Gram Negative bacteria, but the
Gram positive bacteria are not coloured. This leads to the
Identification of whether a bacteria is Gram
Positive/Negative.
4. The Gram reaction reflects the more
complex structure of Gram negative cell
walls
Gram Negative Bacteria have more
chemically complex walls where the
peptidoglycan is supplemented by large
molecules of lipopolysaccharide, which
protects the cell. They do NOT retain the
Crystal Violet stain. Gram negative
bacteria are resistant to penicillin and the
enzyme lysozome.
Gram Positive bacteria lack the complex
lipopolysaccharide layer, and so DO retain
the crystal violet stain. Gram Positive Bacteria are more susceptible to penicillin and the enzyme
lysozome.
5. Microorganisms may be grown in the laboratory if supplied with suitable physical conditions,
nutrients and water.
6. Organisms vary in their requirements and usually grow over a range of temperatures and pH values,
with an optimum within the range.
The Optimum temperature of Bacteria is between 25 and 45, with 37 being the optimum for
mammalian pathogens. The Optimum Temperature is the temperature where the reaction is at its best.
Optimum Temperatures/pH is regulated by Enzymes. If the temperature is too high, the enzyme will
denature, if it is too low the reaction will procede at a very slow rate. In pH the optimum for most
bacteria is slightly Alkaline at 7.4 ( Fungi prefer neutral-slightly acidic range). The Optimum pH is once
again regulated by enzymes. This means that at higher alkalinity or acidity, the enzyme is again
denatured due to shape of the enzyme becoming disfigured. This cannot be reversed.
7. Some organisms are obligate aerobes, requiring oxygen for metabolism, whilst others are obligate
anaerobes and can only survive in the absence of oxygen. Many organisms will grow in either the
presence or absence of oxygen, facultative anaerobes.
3 Types of organisms.
- Obligate Aerobes – Oxygen is required for metabolism, The organism cannot survive if oxygen is
not present.
-Obligate Anaerobes – Cannot grow in the presence of Oxygen
-Facultative Anaerobes – Grow better in Aerobic conditions however can survive in its absence.
8. Nutrients are supplied in nutrient media and include: carbon, usually organic such as glucose;
nitrogen, organic or inorganic; growth factors such as vitamins and mineral salts.
In the laboratory, Nutrients are supplied in nutrient media such as agar. This medium includes CARBON,
usually in the form of GLUCOSE. NITROGEN, in both Organic and Inorganic forms. GOWTH FACTORS such
as VITAMINS and MINERAL SALTS. The Nitrogen us needed for the production of Amino Acids in Protein
Synthesis.
9. Glucose is normally the source of energy.
10. Aseptic techniques involve handling cultures in such a way as to prevent their contamination by
unwanted organisms. However, they have a dual purpose in preventing the contamination of
personnel and the immediate environment by the organisms being cultured.
The aims of Aseptic technique are to prevent contamination to the ENVIRONMENT and contamination
to the SAMPLES.
11. Equipment and media must be sterilised before use by appropriate methods. Heat is commonly
used, examples being the use of an autoclave at a suitable temperature (121oC) for 15minutes, or the
heating an inoculating loop in a Bunsen flame. Heat labile plastics are irradiated. Items must be
protected from contamination after sterilising.
To prevent the contamination of pure cultures by bacteria from the environment
-Sterilise all apparatus and media – prevent initial contamination.
-Use sterile loops & Handle cultures carefully – prevent subsequent contamination
To prevent contamination to the ENVIRONMENT
- Sterilise work surfaces before and after an experiment. - Disinfectant
- Use correct handling techniques
-Hold the Cap with your little finger and NEVER place cap on the bench
-Flame the mouth of the bottle for 2-3 Seconds
-Hold the inoculating loop in the flame until the wire goes red – red hot.
-Lift the lid of the petri dish JUST ENOUGH to allow entry of the inoculating loop
-Secure the lid with 2 pieces of adhesive tape – DO NOT cover with tape as this induces
Anaerobic conditions – Pathogenic Bacteria.
-Incubate at around 25 Degrees
-Do NOT open petri dishes after incubation
Autoclaves = preferred method of sterilisation in a lab
-121 degrees in steam under pressure for 15 minutes
- Ensure that resistant ENDOSPORES are destroyed.
- Plastic Petri dishes can be placed in autoclavable bags and then disposed of afterwards
12. Direct cell counts may be total counts, which include both living and dead cells, and viable counts,
which count living cells only.
Direct cell counts can be divided into
-Total counts – Dead and Alive cells
-Viable counts – Alive cells only
A Haemocytometer may be used. This produces a Total count as dead and alive cells cannot be
differentiated.
Turbidimetry is a method that uses a colorimeter to measure the cloudiness of the culture as cell
numbers are increased.
13. For a viable count a known volume of organisms is added to agar plates, incubated and the
colonies counted. It is assumed that one cell gives rise to one colony. This makes no allowance for
clumping of cells so may cause an underestimate of numbers.
For a viable count, Clumping may account for an underestimation of numbers. The separate colonies
are presumed to have arisen from 1 bacteria. So to find the Total Viable Count, count the number of
colonies present, then times this by the dilution factor to give an estimate number of bacteria.
14. In both cases the original culture usually requires dilution by ten fold steps, serial dilution, in order
to provide a final number within a countable range.
Ten Fold Dilution ( Serial Dilution) is used so that the number of cells that arise when incubated is within
a countable range – not too few or too many.
1cm3 of the original solution is added to 9cm3 of sterile medium and mixed up. Then 1cm3 of this
solution is taken and added to another 9cm3 of sterile medium and mixed up. This is repeated until the
spread of bacteria on the agar plates is in a countable range.
15. A pure culture of an organism is needed for the formation and harvesting of a pure product during
and after growth in a fermenter vessel. The organism must be supplied with suitable conditions for
growth and without competition for maximum efficiency.
16. The vessel should be sterilised beforehand and an appropriate sterile medium used. Filters are
used to prevent contamination through the vessel’s openings. Aseptic conditions and handling are
required to maintain purity.
17. Forced aeration may be needed for maximum growth of aerobes and this aeration may also mix
the culture to improve contact with nutrients. Mixing may be
improved by a separate mixer.
18. Temperature monitoring and control are required to
maintain constant conditions and water jackets remove excess
heat produced during the culture process.
19. Commercially, sophisticated monitors are used to improve
control of temperature and pH, and air inlets may use spargers
or other devices to improve aeration.
20. For the commercial production of penicillin, the fungus
Penicillium notatum is grown in a batch culture and the
antibiotic is produced after the growth phase, when glucose is
depleted. This reflects the need for the organism, when free
living, to reduce competition when food sources are depleted.
Penicillin is produced in a batch fermenter. It takes roughly 30 hours for penicillin production to begin.
Penicillin is secreted by the fungus and accumulates in the medium.
Penicillin is a SECONDARY METABOLITE. This means that it is produced by the fungus AFTER the
exponential phase is complete, when glucose is depleted.
Antibiotic production is a SECONDARY METABOLISM. This means that the antibiotic is produced at a
period in the life of the fungus when there is a change AWAY from it’s OPTIMUM conditions. This
reflects the need for the organism, when free living, to REDUCE COMPETITION when food sources are
depleted.
21. The mycelium is removed by filtration of the culture fluid and the antibiotic purified from the
residual liquid.
After about 6 days, the culture fluid mixture is filtered and penicillin is extracted and purified.