o Staphylococcus aureus could be present/colonise the human
o Incidence of S. aureus in humans:
- persistent carriers; carry one type of strain of the population
- intermittent carriers; form the large part of the population (60%)
- non carriers; never carry S. aureus
What is the role of the MEA plate?
Detection, isolation and enumeration of fungi.
Name the organism that is identified using the Bactistaph.
o A single colony consists of identical cells and can be used to
provide subsequent pure cultures of that species in broth/agar
o The inoculated area forms the primary inoculum.
Explain why agar is used in preference to gelatin.
Agar is from algae.
Gelatin is from animal bones.
Agar is easier to extract and it has a higher melting point.
Why do well-separated colonies appear larger than those in areas of
More nutrients available.
Explain the purpose of passing the dried smear through the Bunsen
Kill the cell, and make it stick to the slide. Alters the cells so it can
readily accept the stains/dyes.
Suggest the effect on the gram stain if a whole colony is used to make
If you use too many cells, you won’t be able to see as well, or even at
all. You want a few cells, so that you can see the cells clearly, not
because the result will be different.
Which structure of the bacterial cell determines its gram reaction?
Peptidoglycan cell wall determines whether the cell wall gets stained
Explain which technical step of the Gram-staining procedure is crucial
in determining the outcome.
Over; + = purple, - = pink
Under; - = pink, + = purple gram + ; retain primary stain (thicker)
gram - ; destain (thinner)
gram + ; violet stain is trapped by peptidoglycan layer which forms the
outer layer of the cell.
gram - ; Outer membrane prevents the stain from reaching the
peptidoglycan layer. The outer membrane is permeabilised by alcohol
and the safarin stain is trapped by the peptidoglycan layer.
o Each bacterial species possesses characteristics by which it can
be fully identified even to subspecies.
o The visible characteristics are termed morphology.
E. Coli Klebsiella sp. P. vulgaris
Gram – rods. They don’t form spores and can live in the
presence/absence of oxygen.
Why do some organisms form clusters and others chains?
Bacteria reproduce rapidly. It is in their nature to split apart when they
reproduce. In reproducing themselves, they have no time to roam
around. Bacteria that multiply quickly and have motility form colonies
Describe the shape and arrangement of endospores. Why are they
difficult to stain?
Stains can’t penetrate the spore covering (exosporium). You have to
heat it to open it than stain it. Also, due to its low permeability and high
degree of resistance due to multiple coats surrounding the spore. We
want to stain them to see if the bacterium cells have highly resistant
spores within their vegetative cells.
What advantage might motility confer on a microorganism?
Bacteria can move toward greater concentration of food, and away
from greater concentration of waste products.
What advantage does the capsule confer on a potential pathogen?
Capsules make the bacteria more resistant to harsh conditions, and self
protect themselves from antibiotics.
o The normal microbial flora of the human body consists of
organisms, which have adapted to the physic-chemical
conditions of their particular habitat.
o Resident flora – where organisms consistently are found at a
given site and multiply. o Resident flora reduces the possibility of invasion by potential
pathogens by occupying attachment sites and successfully
competing for nutrients.
o Opportunistic pathogens – gain access to another site.
o Transient flora – species temporarily found at a given site but
do not multiply there.
o Transient skin flora is of particular significance because they
are frequently implicated in transmission of infection.
o Nosocomial infection – outbreaks of S. aureus in a hospital
environment due to poor hand washing practices.
o Bacterial endospores are recognized as being thermoduric; they
can survive prolonged exposure to 100 degrees.
o Moist heat, using steam at elevated temperature and pressure in
a sealed chamber. The process depends on the steam being
saturated, total removal of air, all surfaces being accessible to
steam and the conditions being maintained for sufficient time.
(121 degrees for 15 minutes at 101 kPa).
What properties of steam make it an ‘ideal’ sterilent?
Rapid, efficient and non-toxic.
It kills cells by denaturing their components resulting in the loss of
function and death.
Moist heat is very efficient as it destroys the microorganisms by
disintegrating their nucleic acids and cell membrane, as well as
denaturing both the enzymes, and the important proteins required for
the microbe to function competently
Compare the biocidal action of moist and dry heat.
The biocidal action of moist heat causes proteins to denature and
coagulation occurs. Whereas, the biocidal action of dry heat dries the
cell up, which causes membrane fusion.
List the various stages that make up the total sterilization cycle.
The sterilization cycle comprises of two stages - the penetration and
holding time. The penetration time ensures that the temperature
required to destroy microorganisms of 121°C is met, while, the holding
time maintains the temperature of 121°C for 15 minutes. If the process
was operated appropriately, it is able to destroy all microorganisms; as
well as bacterial spores that are known to be highly heat resistant.
Explain the term tyndallization.
the process of destroying vegetative bacteria with the use of steam.
Unlike steam sterilization, tyndallization involves the bacteria being
exposed to steam for a total of three times repeatedly and incubating
them for 24 hours between every exposure to steam. Apart from heat, discuss other physical control methods available.
Some physical control methods available are filtration and ultra violet
radiation. Filtration is a method used to reduce the population of
microbes in solutions of materials that are sensitive to heat. It is used to
sterilize different liquids and gases, as well as air. UV Radiation on the
other hand, is used to inhibit the occurrence of replication and
transcription. This method is not as efficient due to its disadvantage of
being a sterilizing agent in limited situations.
Can prions be destroyed by normal steam sterilization process?
Prions are very difficult to destroy as they are resistant to heat, radiation,
as well as many other factors that are capable of destroying a
microorganism. As they are mainly composed of proteins, the normal
sterilization process of 121°C for 15 minutes is not effective against them,
due to the high temperatures required to denature the proteins.
Sterilizing will only work against prions is if the protein is denatured to the
point where they are unable to produce the abnormal folding of
o Microorganisms have optimum requirements for active growth,
but can otherwise tolerate a wide range of conditions.
o The only limits to survival are extremes of temperature, lack of
moisture and the presence of substances that are toxic to the
o 4 environmental influences – temperature, hydrogen ion
concentration, water activity and oxygen availability.
o Differential medium – An indicator system can be incorporated
into the medium to differentiate the desired organism from
others which may also grow.
o Selective media – those using an inhibitory agent to suppress the
growth of unwanted species.
o MacConkey agar is both selective and differential. It is
differential in that the lactose fermenting bacteria appear as red,
pink or purple, and the non-lactose fermenting bacteria
produces colourless and translucent colonies or beige or even
o The media could be made selective by the addition of different
types of bile salts.
o MacConkey agar consists of:
- Nutrient base (2% peptone); peptone = beige/clear colonies
- Bile salts; inhibits all non enteric bacteria but selective for
various enteric bacteria
- Lactose; break down to lactic acid = red/dark purple colonies
- Neutral red indication – pH indicator; detects acid end
products produced by fermentation of lactose lowering the pH, the indicator turns red. Detects ammonia produced by the
deamination of amino acids in peptone raising the pH, the
indicator turns cream/beige colour.
o Enriched media enhance basal media to improve the growth of
the desired organism. This can encourage prolific growth of
unwanted species so selective agents are often included
making an enriched, selective medium.
Explain why you would inoculate the nutrient agar before the
MacConkey agar (w/o salt-NaCl) and then 3 if the same loopful of
culture is used.
MacConkey agar is both selective and differential. It contains bile salts,
and the dye crystal violet, which inhibit the growth of gram + bacteria
and select for gram – bacteria.
Explain why the MacConkey plates should not be incubated for more
than 24 hours.
Bacteria starts utilizing the peptone so we won’t be able to
Discuss the selective and differential basis of the MacConkey agars
used, including the appearance of lac+ and lac- colonies.
lac+ = pink colonies, lac- = beige/yellow.
Basis differentiation – lactose fermentation.
o Quantitative determination of microbial populations is essential
to many lab procedures.
o 3 techniques are widely used – viable count, total cell count and
o Viable count – provides information about the population
density of living cells only.
o Total cell count – counting chamber to obtain an accurate
count of the total number of cells of all types in a known volume
o Turbidometry – uses the level of turbidity measured by
spectrophotometry to estimate the biomass.
o Colony forming unit – possibility that a single colony may have
arisen from two or more bacterial cells in close proximity.
Discuss why duplicate plates of 3 consecutive dilutions are used to
obtain colony counts. Would you always use the same dilutions?
Reduce human errors.
Explain why the viable count is expressed as cfu ml rather than cells ml.
1 cell = 1 colony Why is spread plate method preferable to the pour plate?
Spread plate – easier to count. Volume = o.1 ml
Pour plate – small colonies and embedded in agar.
Why is a washed suspension used to determine the dry mass instead of
an untreated culture?
There’s a fixed amount.
Discuss the major sources of error in this method for determining cell
- dead cells
- extracellular material
o Bacteria are typically grown in batch culture – a close system
with a finite supply of nutrients and oxygen, which will gradually
Predict what would happen if the starting culture which was added to
the growth medium was:
An old culture: An old culture contains old cells where they have
depleted necessary nutrients that are required for division to occur. The
addition of an old culture to a growth medium causes the lag phase to
occur in a growth curve. Time is required for the old cells to recover, as
they may have been damaged previously. Time is also needed for ATP,
essential cofactor and ribosomes synthesis. Furthermore, the previous
medium may have a different composition to the BHI medium, and so,
the cells must produce new enzymes that are suitable for the BHI
In exponential phase: If a culture in exponential phase was added into
a growth medium, an unbalanced growth occurs. This is due to the
difference between the nutrient levels and environmental conditions
from the previous medium to the new growth medium. New suitable
nutrients and cellular constituents must be synthesized until equilibrium
is reached. There are two types of experiments where unbalanced
growth can be observed: shift-up or shift-down. Shift up is essentially
where a culture is transferred from a nutritionally deprived medium, into
a richer medium. While shift-down is where a culture is transferred from
a nutritionally rich medium, into a deprived medium. When a culture is
in a shift-up experiment, it must enter lag phase where they synthesise
new ribosomes and nutrients required for the cells to grow and divide,
before entering the exponential phase where balanced growth is
Suggest reasons to explain the stationary phase.
There are several factors to why microorganisms enter the stationary
phase. When the necessary nutrients become depleted, population
growth decreases. Oxygen availability is another factor. Oxygen is
soluble to a certain extent, thus it has a tendency of becoming depleted rapidly. If this occurs, the cells under the surface are unable
to grow and divide, unless the culture is placed in an aerated
condition. Toxic wastes accumulating may also cause a decrease in
Discuss whether the doubling rate of the organism used is typical of
The mean generation time for the aerated condition is 41.67 minutes,
whereas for the non-aerated condition, it is 39.48 minutes. This indicates
that when Vibrio natriegens doubles much faster