GENERAL MICROBIOLOGY

Summer, 2018-2019
Department of Food Science and Technology
University of Science and Technology of Southern Philippines

EXPERIMENT NO. 1: Preparation and Sterilization of Glassware and Culture

Media
Experimenter: Raizel Eunice F. Tangapa
Date Performed:
Date Submitted:

1. Introduction
Preparation simply means the action or process of making ready or being made
ready for use or consideration and sterilization means the process of making something
free from bacteria or other living microorganism . In Doing laboratories Glassware’s
should be sterilize first before using it to prevent contamination there are types of
sterilization these are the 1. Heated bead sterilization in this process it uses a glass bead
sterilizer. These sterilizers heat to approximately 275-350° C and will destroy bacterial
and fungal spores that maybe found on your instruments. The instruments simply need
to be inserted into the heated glass beads for a period of 10 to 60 sec. The instruments
should then be placed on a rack under the hood to cool until needed 2nd is the Flame
sterilization in this, Instruments can be dipped in ethyl alcohol and flamed to burn off all
bacteria and fungi. Safety is a major concern when using ethyl alcohol. Alcohol is
flammable and if spilled near a flame will cause an instant flash fire. This problem is
compounded in laminar flow hoods due to the strong air currents blown towards the
worker. Fires commonly start when a flamed instrument is thrown back into the alcohol
beaker. In case of fire do not panic. Limiting the supply of oxygen can easily put out fires
the 3rd one is Hot-dry sterilization in this process Metal instruments, glassware, aluminum
foil, etc., can be sterilized by exposure to hot dry air (130°-170°C) for 2-4 hr in a hot-air
oven. All items should be sealed before sterilization but not in paper, as it decomposes
at170°C and the 4rth and lastly is Autoclaving. Autoclaving is a method of sterilizing with
water vapor under pressure. Nearly all microbes are killed by exposure to the super-
heated steam of an autoclave for 10-15 minutes. All objects should be sterilized at 121°C
and 15 psi for 15-20 min. Cotton plugs, gauze, labware, plastic caps, glassware, filters,
pipettes, water, and nutrient media can all be sterilized by autoclaving, but autoclaving is
not advisable for metal instruments due to the rust it may cause. Then in this activity
cultured media is prepared so Why is culture media prepared? Enrichment media contain
specific growth factors that allow the growth of metabolically fastidious microorganisms.
An enrichment culture is obtained with selected media and incubation conditions to
isolate the microorganisms of interest. Culture media preparation is one of the routine
tasks common to many microbiology laboratories. This is true in the food industry, where
producers regularly monitor food and environmental samples for spoilage and pathogenic
microbes as an early indication of breakdown in processing hygiene. It might be routine,
but did you know that there are key steps to follow for success in culture media
preparation? The way you handle your culture media—from storing it before
preparation, to lifting the agar plate lid, to incubation—affects your results. Selective
media cannot select for pathogens if the starting materials are poor quality, compromised
or contaminated.
2. Materials and Methods

2.1. Materials:
Food Sample Culture Media: NA, PDA,
Peptone Water Petri dishes
Alcohol lamp Pipettes
Cotton plugs Test tubes
Aluminum foil Graduated cylinder
Glass stirring rod Analytical Balance
Erlenmeyer Flasks

2.2. Methods:

Preparation of Cotton plugs

1. Cut cotton sheet and measure to fit in glassware opening.
2. Wrap with bandage gauze and tie.

Preparation and Sterilization of Glassware:

1. Wash glassware with detergent and water. Liquid detergents are preferred since it is
easily rinse off and leave no water beads on the glass. Rinse with distilled water.
2. Dry glassware at room temperature.
3. For preparation and sterilization of petri plates, wrap each pair in aluminum foil,
make sure it is properly covered and air pockets are eliminated that may cause
insufficient sterilization.
4. To prepare pipettes for sterilization, wrap pipettes in aluminum foil by two’s. Make
sure it is properly covered.
5. To prepare test tube and flasks for sterilization, plug test tube and flask mouth with
cotton plugs, make sure cotton plugs are not too tight nor too small for the opening.
Cover cotton plugs with aluminum foil. For screw capped tubes, loosen the caps before
sterilization, but not too loose for it to fall off.
6. Sterilize glassware at 15psi for 15 minutes.

Preparation and Sterilization of Culture Media

1. Weigh the ingredients for culture media: Nutrient Agar, Potato Dextrose, and
buffered peptone water.
2. Mix the ingredients in 500mL distilled water for Nutrient Agar and Potato Dextrose
Agar, and 200mL distilled water for buffered peptone. 3. Dissolve the ingredients by
heating in a water bath with constant stirring. 4. Transfer in a clean glass container and
sterilize at 121ºC for 15minutes.
3. Results and Discussion

1. What are the functions of each culture medium used?

The Preservation Culture Media. This is composed of all the basic nutrients required for
a microbial growth and is used to preserve a specific type of microorganism, preferably
bacteria or a set of different microbial entities for a long period of time. These are some
culture media and its functions 1st is the The Preservation Culture Media This is composed
of all the basic nutrients required for a microbial growth and is used to preserve a specific
type of microorganism, preferably bacteria or a set of different microbial entities for a long
period of time.The basic purpose of this culture is to let these microorganisms grow safely
in an ensured environment that has all the important nutrients and to protect them against
any environmental damage so these organisms can be used when needed. 2nd is The
Enrichment Culture Media this is a liquid medium which allows the microorganisms to
multiply and has the essential nutrients that are required for it.It is usually composed of
bacteria taken from a liquid source such as pond water. The basic nutrient broth is the most
commonly used 3rd the Selective Culture Media This is a special type of media which allows
the growth of certain microorganisms while inhibits the growth of the others.For example
if we want to isolate a specific bacteria let’s say that can with stand an acidic environment
from a sample of pond water and get rid of others, a selective media with a low pH will be
taken which will allow the growth of only those organisms that can withstand acidity and
will kill the others that cannot. 4th is the Differential Culture Media This is a media that is
used for differentiating between bacteria by using an identification marker for a specific
type of microorganism.The selective and differential culture media are opposites to each
other in a way that one inhibits the growth of other organisms while allowing the growth of
some while the other does not kill the others but only highlights one type so that was some
of the culture medium used and their function.

2. What is the purpose in sterilizing glassware and culture medium?

In sterilizing glassware and culture medium t does not prevent contamination, but slows
its growth. ... It is much better to store media at room temperature and detect contamination
before the medium is used. (1) Sterilizing With Moist Heat. Moist heat provided by an autoclave
or pressure cooker is an efficient way to sterilize most materials. The purpose of culture medium
to be sterilize is Autoclaves operate at high temperature and pressure in order to kill
microorganisms and spores. They are used to decontaminate certain biological waste and
sterilize media, instruments and lab ware and when sterilizing glassware such as bottles, petri
dishes and test tubes, dry heat is required and this is carried out in a hot air oven. The ideal
temperature of the oven needs to reach is at least 160°C and the contents need to be regulated
at this heat for 45 to 60 minutes.

3. What is the advantage of using autoclave rather than using pressure cooker?

A very basic autoclave is similar to a pressure cooker; both use the power of steam to
kill bacteria, spores and germs resistant to boiling water and powerful detergents. The most
advanced autoclave cannot be compared to a pressure cooker though their basic functions are
similar. Autoclaves use pressurized steam as their sterilization agent. The basic concept of an
autoclave is to have each item sterilized -whether it is a liquid, plastic ware, or glassware- come
in direct contact with steam at a specific temperature and pressure for a specific amount of time
while A pressure cooker is needed to sterilize your substrates and casing material. Also, some
supplies like scalpels can be sterilized in a pressure cooker. When it comes to sterility there is
only one rule: Or it is sterile or it is non-sterile, there is no in-between After all, one of the most
important benefits of steam autoclave sterilization is that it requires considerably less time and
heat than a dry heat sterilizer, due to steam's capacity to transfer energy.

4. Conclusion

The preparation of culture media and their underlying culture technique goes hand in
hand and affect each other in either a good or bad way. When the preparation of a culture media
is already poor that is, it was not sterilized properly or that the experimenter made some form
of mistake on the culture process, even though the culture technique is good, the output or the
growth of bacteria would be influenced by the quality of media used. For the culture technique,
it goes to say that if the culture technique was executed in an unsatisfactory manner then
microbial growth would still be affected. This case is exemplified by our plate sample, our
streaking was relatively poor thus it had caused unfavorable results. Thus proper and correct
way of streaking needs to be enhanced in the future so that results would be promising. Overall,
correlation between the culture median and the culture technique was emphasized on the
experiment, also the importance of correct way of executing or conducting this activity is given
highlight in the experiment.

5. Bibliography

Oppsi Suguoi (2017) Preparation and Sterilization of Culture Media and Glasswares. Retrieved: May
11, 2019. From
https://www.academia.edu/31427064/Preparation_and_Sterilization_of_Culture_Media_and_
Glass_wares

Re eren&es:%ttp:@@z' aabioproses10.blo$spot.&om@2011@0D@lab>D>preparation>and
>sterilization>o .%tml
2017
6. Appendices
 GENERAL MICROBIOLOGY
Summer, 2018-2019
Department of Food Science and Technology
University of Science and Technology of Southern Philippines

EXPERIMENT NO.2: Serial Dilution of Selected Food Samples

Experimenter: Raizel Eunice F. Tangapa
Date Performed:
Date Submitted:

1. Introduction

A serial dilution is the stepwise dilution of a substance in solution. Usually the

dilution factor at each step is constant, resulting in a geometric progression of the
concentration in a logarithmic fashion. Making a 10 Fold Dilution. The first step in
making a serial dilution is to take a known volume (usually 1ml) of stock and place it
into a known volume of distilled water (usually 9ml). This produces 10ml of the dilute
solution. The objective of the serial dilution method is to estimate the concentration
(number of colonies, organisms, bacteria, or viruses) of an unknown sample by
counting the number of colonies cultured from serial dilutions of the sample, and then
back track the measured counts to the unknown concentration. A serial dilution is a
series of sequential dilutions used to reduce a dense culture of cells to a more usable
concentration. Each dilution will reduce the concentration of bacteria by a specific
amount. Serial dilution techniques are routinely used in hospitals, public health,
virology, immunology, microbiology, pharmaceutical industry, and food protection
(American Public Health Association, 2005, Hollinger, 1993, Taswell, 1984, Lin and
Stephenson, 1998) for microorganisms that can grow on bacteriological media and
develop into colonies. A list of bacteria that are viable but nonculturable (VBNC), the
detection of such microorganisms, and the process of resuscitation of cells from
VBNC state are addressed by Oliver, 2005, Oliver, 2010. In the work presented here it
is assumed that the microorganisms are culturable.
2. Materials and Methods

2.1. Materials:
Food Sample Culture Media: NA, PDA,
Peptone Water Petri dishes
Alcohol lamp Pipettes
Cotton plugs Test tubes

2.2. Methods:

1. Mix 1-gram of food sample with 9mL buffered peptone water.

2. Perform serial dilutions of the sample from 10-1 to 10-3.
3. Prepare duplicate plates for each appropriate dilution and pipette 1mL of food sample.
4. Pour 15-20mL of culture media per plate for the isolation of common bacteria (NA), yeasts
and molds (PDA).
5. Allow the agar to solidify and incubate plates upside down at 30ºC for 1-2 days for bacteria
and yeasts; and 2-3 days for molds.
6. Count the colonies on plates containing 30-300 colonies for bacteria and yeast or 25250
colonies for molds. Report counts as colony forming unites/mL or gram (CFY/mL or g)
sample.

3. Results and Discussion

Observations
1. Observe the growth characteristics of each plate dilution. Report the following:
a. Bacteria
i. Growth characteristics – whether smooth, rough, mucoid, etc
ii.Pigment formation – both the colony and color change of the medium
iii. Form of growth – punctiform, circular, irregular
b. Yeast
i. Surface growth – colony may either be smooth or rough
ii. Pigment formation – color change for both colony and agar medium
c. Molds
i. Growth characteristics – whether submerged, aerial, fluffy or cottony
ii.Color change of the medium (if any)
iii. Color of the fruiting body

Table 1. Estimated number of microorganisms in selected food samples.

Number of colonies (CFU/g or mL) plate count (ESPC)
Microorganism -1 -2 -3
10 10 10
Nutrient Agar R1- 124 R1- 35 R1- 16
(NA) R2- 80 R2- 184 R2- 24
Potato Dextrose R1- 5 R1- 11 R1- 36
Agar (PDA) R2- 8 R2- 12 R2- 27

Table 2. Cultural characteristics of each dilution.

Microorganism Form Elevation Margin Color
 Bacteria 1
Bacteria 2
Yeast 1
Mold 1

1. Why is it that plate count method is used in the enumeration of microorganisms in food samples?
What are its advantages and disadvantages over other methods of enumeration?

The number of bacteria in a given sample is usually too great to be counted directly. However, if the
sample is serially diluted (see Fig. 7) and then plated out on an agar surface in such a manner that
single isolated bacteria form visible isolated colonies (see Fig. 1), the number of colonies can be used
as a measure of the number of viable (living) cells in that known dilution. However, keep in mind that
if the organism normally forms multiple cell arrangements, such as chains, the colony-forming unit
may consist of a chain of bacteria rather than a single bacterium. In addition, some of the bacteria
may be clumped together. Therefore, when doing the plate count technique, we generally say we
are determining the number of Colony-Forming Units (CFUs) in that known dilution. By extrapolation,
this number can in turn be used to calculate the number of CFUs in the original sample. Normally,
the bacterial sample is diluted by factors of 10 and plated on agar. After incubation, the number of
colonies on a dilution plate showing between 30 and 300 colonies (see Fig. 1) is determined. A plate
having 30-300 colonies is chosen because this range is considered statistically significant. If there are
less than 30 colonies on the plate, small errors in dilution technique or the presence of a few
contaminants will have a drastic effect on the final count. Likewise, if there are more than 300
colonies on the plate, there will be poor isolation and colonies will have grown together. Advantages
of sampling over complete enumeration 1st it Reduced cost and enlarged scope. Sampling involves
the collection of data on smaller number of units in comparison to the complete enumeration, so the
cost involved in the collection of information is reduced.

2. What is the purpose of inverting the plates during incubation period?

Plates are incubated upside down (agar up), so that condensation does not drip onto the plate and
interfere with the developing microbes .Petri plates are incubated upside-down to lessen the risk of
contamination from airborne particles settling on them and to prevent the accumulation of any
water condensation that may otherwise disturb or compromise a culture. And the main reason why
petri dishes are inverted during incubation to prevent condensation from falling into the microbes,
thereby contaminating samples. Condensation in Petri dishes causes bacterial samples to spread and
potentially mix with each other. Warm incubators tend to attract more condensation, so the dishes
are inverted
 4. Conclusion
By doing the serial delution I therefore conclude that when performing multiple
serial dilutions, with greatly increased your skill on manual pipetting and broaden your
knowledge on how many counts is there In possible in the 1st, 2nd or 3rd tube if it will increase
from the -1 or it will decrease.

Appendices