Microbiology Practical 2: Dry heat sterilization of Talc and determining the number of

colonies that have formed after dry heat sterilization.

Aim:
To determine the number of colony forming units after dry heat sterilisation.
Introduction:
Sterilization is a process where all the living microorganisms, including bacterial spores are
killed. Sterilization can be achieved by physical, chemical and physiochemical means.
Including dry heat sterilization, which is a physical means of sterilization (Environmental
Health and Safety, 2011).

Sterilizing by dry heat is accomplished by conduction. The heat is absorbed by the outside
surface of the item, then passes towards the centre of the item, layer by layer (Hendrix.edu,
2012). The entire item will eventually reach the temperature required for sterilization to take
place. Dry heat does most of the damage by oxidizing molecules. The important cell
constituents are destroyed and the organism dies (NCBI, 2012).

Dry heat sterilization has many advantages. It is relatively easy to install and is low
maintenance and cost. This is particularly important for many areas which have limited funds
such as Grahamstown. This process and non-toxic and is environmentally friendly. It is noncorrosive for metal and sharp instruments, which is very important in a lab setting
(Environmental Health and Safety, 2011).

Dry heat sterilization can only be used for thermo stable, moisture sensitive or moisture
impermeable pharmaceutical substances (Prior Clave North America, 2014). These include
products like; Dry powdered drugs, Suspensions of drug in non aqueous solvents, Oils, fats
waxes, soft hard paraffin silicone, Oily injections, implants, ophthalmic ointments and
ointment bases etc. Including Kaolin and Talc (Acharya, 2013).
Talc (H2Mg3O12Si14) has a molecular weight of 379.26 g/mol. It is an odourless white to
grayish white very fine crystalline powder. Which readily adheres to the skin. It is non-toxic
and non-flammable. It is used as an anti-caking agent to improve powder flow in tablet
compression. Talc is used cosmetically in talcum and baby powder as an adsorbent
(Magnesium Silicate, 2016).
Kaolinite (Al2O5Si) has a molecular weight of 162.045577 g/mol. Its appearance ranges from
white, brownish white, greyish white, yellowish white to greyish green. It has an earthy lustre
and the triclinic crystals are translucent. It is used in industry as adsorbents, fillers,
intermediates and absorbents, processing aids, specific to petroleum production. It is used
commercially as automotive care products, building/construction materials not covered
elsewhere, Metal Products not covered elsewhere and water treatment products
(Pubchem,2014).
Acetone (C3H6O) has a molecular weight of 58.07914 g/mol. It is a colourless, volatile,
flammable organic liquid solvent that has a characteristic taste and smell. It is miscible in

water. It should be stored in a closed fireproof container away from heat, sparks, and flames
and away from drain or sewer access (HiMedia Publications,2013).
Spread plating was used to plate the Petri dishes with the substances. In order to count the
number of bacteria in a sample, this is a method that allows an even growth of bacterial
colonies. The spread plate technique creates an even distribution of bacteria on an agar plate
(Acharya, 2013).

We were given sample number 22 and 37 and therefore had to decide which sample was
sterilized before the dry heat sterilization.

2. Methods and Materials

2.1. Pre-practical and safety procedures
All safety clothing was put on. This included gloves, protective glasses and a lab coat to
protect the skin and eyes from contact with the samples that were given. The samples were
prepared using the dry heat sterilization of talc. We were given three samples to plate which
were prepared using sterilized and unsterilized talc. The third sample served as a control.

2.2. Sterilization
A sterile environment was created by wiping the bench tops with 70 percent ethanol to kill
any microorganisms present. The ethanol penetrates the cell wall, killing single celled
microorganisms by breaking up the protein structures. The Bunsen burners were then lit with
matches to create a sterile area under which all procedures were carried out to prevent
contamination to any of the samples that were plated. This was carried out to ensure that valid
results were obtained from the procedure and other microorganisms did not enter and alter the
final results.

2.3. Plating of samples

The samples were then plated under the Bunsen burners. The inoculating syringe was covered
with a disposable cap to prevent cross contamination of samples. 0.1ml of solution was
removed from the first sample flask (number 27) and inoculated into a plate that contained a
given amount of nutrient agar. The glass spreading rod (cell spreader) was sterilized by
rinsing it in 70 percent ethanol and passing it through a flame. The cell spreader was then
used to spread the contents of the petri dish uniformly while the plate was being turned. This
procedure was repeated for the second sample flask (number 33) and the third flask that
served as the control. Each sample was plated twice which produced 6 plated samples.

2.4. Incubation
The plates were transferred from the sterile area on the bench top to the incubating oven
located in the labs. The plates were incubated for a 24-hour period after which they were
inspected and the results of the plating was obtained.

RESULTS

Figure 1: Control Sample,A (left) and B (right)

Figure 2: Control Sample A

Figure 4: Sample 22, A (left) and B (right)

Figure 3: Control Sample B

Figure 5: Sample 22 A

Figure 7: Sample 37, A (left) and B (right)

Figure 6: Sample 22 B

Figure 8: Sample 37 A

Figure 9: Sample 37 B

The colonies above were white; it was slightly visible to the naked eye. Its exact shape could
not be determined, as one would need a microscope.
Table showing a summary of results
Growth
Number of

CFU Value

Contaminated

Can not be

?
A- No

Figure 1

No growth

Colonies
0

Figure 3

Growth

Too many to

calculated
Can not be

B- Yes
A- Yes

Figure 7

Growth

count
Too many to

calculated
Can not be

B- Yes
A- Yes

count

calculated

B- Yes

Figure 7 is more contaminated than Figure 3 i.e Number 37 was more contaminated than
number 22.
Discussion
According to the above results, number 22 was the sterilized sample. Since number 37 had
more bacterial growth. However, since number 22 was sterilized, colonies should not have
grown and this could be attributed to a number of reasons.
The purpose of dry heat sterilization is to destroy any possible bacterial growth that could
have occurred. Working on the principle that after a certain high temperature damage to the
microorganisms would incur damage via oxidation. (NCBI, 2012)The samples we were given
were made up of a control sample containing no bacteria and two unknown samples, one
contaminated and one not. All three samples were then exposed to dry heat to destroy any
possible bacteria (CDC, 2014).
Upon analysing the results, all of the samples had a very large bacterial growth to the extent
that a CFU value could not be calculated. The samples were not diluted and hence we got

such a large growth. If the samples had been diluted, we would have gotten better results as
we could have actually counted the colonies and have gotten a CFU value.
This may have also occurred if cross contamination had occurred (NCBI, 2012).
Contamination of the samples clearly did occur because the control was contaminated as
shown in figure 1 b.
There are multiple points of cross contamination to occur. Incorrect sterilization of the
hockey stick for spread platting could lead to bacteria from the contaminated sample entering
other samples. This could have occurred due to using too little ethanol and exposing it to the
flame for a short period of time. The agar spread plates may have been open for too long and
may have been at too great a distance to maintain a sterile environment. Contamination may
also have occurred in the oven as there was continues opening and closing of the doors. The
broth may also have contained a too concentrated amount of the bacteria. It was probably a
culmination of the above reasons.
The practical was conducted in an open environment as the sterile laboratory was too small to
accommodate us as a class.
The practical could have been improved by ensuring the hockey stick was sterile by
increasing the exposure to a flame and to the ethanol. The environment should also have
been thoroughly sterilized with the ethanol as well as ensuring the spread plates were open
for as short a time as possible.

Conclusion:
The results we obtained were not what we expected. This was attributed to a number of
reasons, care should be taken when conducting the experiment for the second time.
Dry heat sterilization is an excellent technique if done properly and in a sterile environment.

References

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