EXPERIMENT 7 : MICROBIAL PRESERVATION

TECHNIQUES
1.0 SUMMARY

The microbial preservation techniques were used in this experiment. This

technique was used to preserve microorganism and control culture in the standard method of
analysis. The purpose of this experiment was to study the technique of glycerol preservation.
In this experiment, the solution of 30% glycerol was prepared by mixing 30 ml of glycerol
with 70 ml of water. The solution was transferred into a universal bottle and sterilize by
autoclaving at 121C for 15 min. 500 l of sterile 30% glycerol was aliquot into sterile micro
centrifuge tube. The culture media was labelled and was placed in the freeze storage at 4C.
The small volume of cells was transferred to an agar plate and streak. The viability of the
bacterial culture was checked after 2 weeks by inoculating the cells on Nutrient agar plate.
The viability of the culture was checked after 2 weeks of incubation at 37 C. The result in
Figure 1 of this experiment shows that there is no growth of microorganism on nutrient agar
plate during the period of incubation. Based on the discussion there were error occurred
during preparation on the stock of glycerol solution. The problem occurred was on the
temperature time and location. These three factors have been affected on the microorganism
to growth on the agar plate. The end point of our analysis and observation shows that the
preservative techniques was achieved on the objective of this experiment which is using to
the glycerol solution but it failed to growth on the nutrient agar plate. It was the techniques
that can be practices and gain knowledge to control culture in the standard method and
handling it in the future field of industry.

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2.0 OBJECTIVE
1. To study microbial preservation in which is glycerol preservation.
2. To study the technique of glycerol preservation.
2.0 INTRODUCTION
Bacteria can survive for a short period of time at 4C. Cultures grown on agar slants
or plates that are used daily or weekly can be stored in a refrigerator assuming that precaution
has been taken to avoid contamination. Cultures should be prepared using standard
techniques and then sealed before storing. It is recommended using screw capped tubes for
slants while for cultures on Petri dishes, the plates need to be sealed with Parafilm. This is
because it helps to prevent molds from sneaking into the plates and it slows the agar from
drying. For anything over a week or two, cultures can be stored as stabs in small, flatbottomed screw capped vials and microfuge tube.
Freezing is a good way to store bacteria. Generally, the lower the storage
temperature, the longer the culture will remain the originality. Freezers can be divided into
three categories which are laboratory, ultralow, and cryogenic. However, the problem that
may be faced by the bacteria stored in freezers is ice crystals. The bacteria can be damaged
by dehydration caused by localized increases in salt concentration. Solutes accumulate in the
residual free water as water is converted to ice and this high concentration of solutes can
denature biomolecules. Ice can also rupture membranes such as cultured animal cells. To
overcome this problem, glycerol is often used as a cryoprotectant to lessen the negative
effects of freezing. By adding glycerol to final concentration of 30% with bacteria will help
to keep cells viable under all freezing conditions.
Laboratory freezers can pull temperatures down to -20 to -40C. These are single
stage systems (one compressor) and often called general purpose freezers. Bacteria can be
stored for moderate periods of time, in general purpose freezers. It is best to use freezers
without frost-free temperature cycling because it can wreak havoc on cells and other
temperature sensitive biomolecules. General purpose freezers are inexpensive and found in
most labs, thus the freezers are readily available for storing cultures. The downside is that it
is not sufficiently cold for long-term storage.

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3.0 PROCEDURE
1. A solution of 30% glycerol (v/v) was prepared by mixing 30 ml of glycerol with 70
ml of water. The solution was transferred into a universal bottle and sterilize by
autoclaving at 121C for 15 min.
2. 500 l of sterile 30% glycerol was aliquoted into sterile microfuge tube.
3. 500 l of bacterial culture was added to the tube and mix with the glycerol using a
vortex mixer.
4. The tube was labelled with the organism name, strain, date, etc.
5. The tube was placed in the freezer (-20 C) and record its location.
6. Only a small volume of culture needs to be removed from the tube. The tube doesn't
need to be thawed to activate the bacteria. The tube was opened and scraped the
frozen culture with a sterile pipette tip. The tube was replaced into the freezer (-20
C) immediately. The small volume of frozen/thawed cells was transferred to an agar
plate and streak. The thawed culture was disposed appropriately. The viability of the
bacterial culture was checked after 2 weeks by inoculating the thawed cells on
Nutrient agar plate.
7. The viability of the culture was checked after 24 hours of incubation at 37 C.

EXPERIMENT 7 : MICROBIAL PRESERVATION

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4.0 RESULTS

Figure 1: No microorganisms observed on the nutrient agar plate

EXPERIMENT 7 : MICROBIAL PRESERVATION

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4.0 DISCUSSION

This experiment was the preservation techniques of microbial culture which is

used to preserve microorganisms and for control culture in standard method of analysis. The
purpose of this experiment was to study on the preservation techniques by using the glycerol
solution. These techniques were used on the frozen storage at 4C. They were used 30% of
glycerol solution which is containing 30 ml of glycerol and 70 ml of water.

In this solution they were mix with the bacteria culture of E.coli. This was aliquot
in the sterile micro centrifuge tube and was store at 4C. Then, for the activation of bacteria, a
small volume of culture was removed from the tube. The result from figure 1 was divided
into four parts of solution by labelling the name on each of student on the nutrient agar plate.

According to the result after two weeks culture bacteria of E.coli on nutrient agar
plate, it shows that there is no bacteria culture on the nutrient agar plate because there was an
error occurred during this experiment. The solution of glycerol that has been store at 4C was
not clearly on the set of temperature and the solution was not frozen and not in solidity
condition. This method also might store in the long period of time. This matter can kill the
microbes because of the toxic products that can get accumulated and give an effect on
bacteria to growth on the agar plate.

There was also might be because of the streaking technique. It is because of the
materials of hot loop that might kill the sensitive microbe in the plate. This error also might
be occur when using the wrong technique of streaking method or taken in a small amount of
bacteria, which bacteria cannot be growth in the plate. The end point of our analysis and
observation shows that the preservative techniques have been used during this experiment
which has been achieved on the objective of this experiment but it failed to growth on the
nutrient agar plate by using glycerol solution after 2 weeks of observation.

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5.0 CONCLUSION

In this experiment, the microbial preservation techniques were used. Based on the
result the culture on the bacteria of E.coli which is mix with the storage solution of glycerol
has been observed on the nutrient agar plate. The solution has been stored at 4C. The culture
media have been incubating in 2 weeks. It shows that there is no microorganism growth on
the nutrient agar plate. According to the discussion, they were state that there is an error
occurred during the experiment. The storage of the solution was having a problem during on
the storage period. The set of the temperature might be change or not properly check on the
temperature before store the solution. In this matter, the temperature should be set on the right
temperature and always checked before place the solution whether it was in the right
condition. It can give the solution in the good condition and it not be damaged. There also
might be in the long period of time. This can be an error for the microorganism to growth on
the agar plate because the toxic products that can get accumulated and kill the microbes. In
this situation, the period of time should be managed properly. This matter can be reduced by
conduct with one or two person to keep on checking twice a week. There also might be a
problem when using the streaking method. It can be on the materials of hot loop that might
kill the sensitive microbe in the plate. This matter is also important to this process in this
experiment. This condition is the process that determines the bacteria growth. There also have
a way to prevent the bacteria from contaminated or been damaged which is sealed the culture
media during the incubation period. Lastly, this technique was used to preserve microbe and
give the knowledge to work properly during to examine this process.

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6.0 TUTORIAL

1. Discuss microbial preservation techniques besides glycerol stock preparation.

The microbial preservation techniques is by freeze drying. Bacteria can be freezedried by suspending log-phase cells in a lyophilisation medium and then freezedrying the suspension. Not all bacteria can be successfully freeze-dried. 6-8 certain
strains might not survive the process or die rapidly once freeze-dried. The best way to
determine if a strain is amenable to freeze-drying is to empirically evaluate its
stability postfreeze-drying while maintaining a live culture as a backup. Once freezedried, it is best to store the bacteria at or below 4C. Another technique is by
cryopreservation. Cryopreservation is the use of very low temperatures to preserve
structurally intact living cells and tissues. Unprotected freezing is normally lethal and
this chapter seeks to analyse some of the mechanisms involved and to show how
cooling can be used to produce stable conditions that preserve life. The biological
effects of cooling are dominated by the freezing of water, which results in the
concentration of the solutes that are dissolved in the remaining liquid phase. Rival
theories of freezing injury have envisaged either that ice crystals pierce or tease apart
the cells, destroying them by direct mechanical action, or that damage is from
secondary effects via changes in the composition of the liquid phase. Cryoprotectants,
simply by increasing the total concentration of all solutes in the system, reduce the
amount of ice formed at any given temperature; but to be biologically acceptable they
must be able to penetrate into the cells and have low toxicity.

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2. Propose a laboratory test to a food manufacturing company to determine the

microbiological status of a food preparation surface.
a. Sampling sites and surface selections.
Conduct a microbial survey at the food company. The facilities were selected at
random from everywhere in the area. Each facilities was tested twice monthly over
the course of an 8-month period for a total of 16 sampling periods per centre. Four
areas within the facilities were sampled: one pilot plant area, one packaging area, and
two food preparation areas (two separate locations in the food preparation area or
kitchen). All surfaces were assayed three times daily, including preopening (prior to
6:30 a.m.), during lunch (11:00 a.m. to ca. 1:00 p.m.), and following final snack time
and clean-up of the day (ca. 3:00 p.m. to 4:30 p.m.), to monitor the microbiological
quality of each surface throughout the day.
b. Sample preparation for microbiological analysis.
The sampling area was delineated with a sterile stainless steel template which exposed
a surface area of 50 cm. Sampling was performed by swabbing the area in
accordance with the manufacturer's instructions. After sampling, the swabs were
marked with an identification code and placed in food storage bags in an insulated
tote Samples were stored at 4C until testing, and all samples were analysed within 1
hour of arrival at the laboratory.
c. Microbiological counts.
Each swab solution (1 ml) was plated on aerobic count agar plates, and plates were
incubated at 32C for 48 h. Upon the completion of incubation, plates were counted
on standard colony counter. Sampling procedures for Escherichia coli. After surface
swabbing was completed, 1 ml of swab solution was plated and incubated at 32C for
24 to 48 hours according to the manufacturer's instructions on E. coliagar plates.
Results for all counts were reported as the number of CFU per 50 cm.

7.0 REFERENCES

EXPERIMENT 7 : MICROBIAL PRESERVATION

TECHNIQUES
1. Old, D.C.; Duguid, J.P. (1970). "Selective Outgrowth of Fimbriate Bacteria in Static
Liquid Medium". Journal of Bacteriology (American Society for
Microbiology)103 (2): 447456.
2. Madigan, Michael T. (2012). Brock biology of microorganisms (13th ed.). San
Francisco: Benjamin Cummings.
3. Diaz E (editor). (2008). Microbial Biodegradation: Genomics and Molecular Biology
(1st ed.). Caister Academic Press.
4. Horneck G (1981). "Survival of microorganisms in space: a review". Adv Space Res 1
(14): 3948. doi:10.1016/0273-1177(81)90241-6.
5. Zerek, B. F. (n.d.). The preservation and protection of library collections: A practical
guide to microbiological controls. doi:How to preserve microorganism.