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Microbiology Laboratory

Lab 1

Aseptic Technique and Media Preparation

Introduction:

The media for culturing desirable microorganisms will readily grow undesirable
contaminants, especially molds and other types of fungus, and bacteria from the user’s
skin and hair. It is therefore essential to protect cultures from contamination from
airborne spores and living microorganisms, surface contaminants that may be on the
instruments, and from skin contact. Bacteria and other contaminants cannot fly. Nearly
all forms of contamination are carried on microscopic dust particles that make their way
onto sterile surfaces when they are carelessly handled.

Aseptic technique refers to a procedure that is performed under sterile

conditions. It is a method used to prevent contamination microbial contamination of
themselves, which may result in infection, contamination of the environment they are
working in (e.g. fomites), and contamination of the specimen they are working on, which
is especially important when a pure culture is desired. It includes techniques using flame
sterilization. It is used whenever specimens are to be transferred between media, for
example, when subculturing.

Objectives:

Upon completion of the experiment, student should be able to:

1. Demonstrate aseptic techniques/procedure before performing bioprocess

experimental work.
2. Compare the different on cultivation nutrient when handling under aseptic
techniques and under normal environment workplace.
3. Prepared the nutrient according to the formulation and requirement.

Experimental

a) Aseptic Techniques Procedure

Aseptic technique procedures below involved with transferring of the sample

(subculturing) and utilization of laminar flow cabinet.

1) For most bacterial cultures, sterile loop or needle will be used to inoculate or to
obtain an inoculum.

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2) Flame a loop or needle to red-hot just prior to use, burning off any organic
material. Do not allow the loop to touch anything except the specimen itself, until
the entire procedure is finished.

3) Cool the instrument by touching the sterile agar or liquid surface prior to touching
a culture (or else you will kill it).
4) Preparing to transfer the specimen, hold the tube or flask in one hand, typically
the opposite of the writing hand. Then open the tube or flask containing the
specimen source and briefly hold the top of it in the flame, to kill unwanted
microbes. Pass the neck of the tube or flask with sterile contents through a flame
before taking off the cap. Hold the cap with opening down and the tube horizontal
or nearly so. Convection from the heated neck will prevent dust from falling into
the opening.
5) Use the inoculating loop with the writing hand to retrieve the specimen, and then
sterilize the top of the tube or flask again before immediately closing it to
minimize the possible time for contamination of the specimen in the source tube
or flask.
6) Pass the neck of the tube or flask with a culture through a flame before putting
back the cap.
7) Re-sterilize the instrument after performing the procedure, putting down safely
without burning the bench, yourself, or another student. Keeping in mind that the
specimen on the inoculating loop could be contaminated during every moment it
is exposed. Repeat the previous step identically with the tube or flask in which
the specimen is to be deposited.
8) Use sterile disposable pipettes to remove samples from a broth culture that must
be kept uncontaminated.

Never leave a culture dish open, even for a short time when viewing colonies of
organisms, unless you intend to destroy it. When it is necessary to open a dish, keep the
lid close to the dish, open it only as far and as long as is necessary to accomplish the
procedure, and keep the lid between your face (and your germs!) and the agar surface.
Always be aware of where your hands are, where your face is, and whether or not your
culture is in a position to be contaminated. If you have long hair, make sure it does not
hang into your plate. Hair is full of potential contaminants, and is one of the principle
sources of contaminating microorganisms. If you have an open flame, long hair that is
not tied back or loose clothing can be hazardous to your health. Keep flammables away
from the flames, including alcohol used for sterilizing instruments; do not place a heated
loop or glass rod into an alcohol dish.

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A contaminated culture can often be rescued; however, there is always the risk that
you will re-isolate the wrong microorganism. Besides, you do not have that kind of time
to waste. Exercise extreme care to keep your cultures pure.

Sterile Cabinet

A simple laminar flow hood protects exposed sterile surfaces that are placed inside. A
containment hood does both jobs, keeping airborne particulate matter from going in or
out. To use a hood properly, remember these points.

 Keep all surfaces clean and dry. Clean the surfaces with ethanol.
 Frequently use the UV light to sterilize the interior surfaces. Ensure that the door
of the laminar flow is closed before the UV light is switch on. Do not stare at the
light, which can cause retinal damage.
 The opening must not exceed the recommended sash height.
 Surfaces kept to back of the hood are more likely to remain sterile, as are objects
kept close to the table surface.
 Keep non-sterile objects closer to the front, sterile objects to the back.
 Keep the hood uncluttered.
 Never reach over a sterile surface - you WILL contaminate it; reach around sterile
surfaces if necessary.
 Watch for long hair hanging over sterile surfaces.
 Place lids with sterile side DOWN; do not turn lids upside down; nothing will jump
up and contaminate the lid.
 Use slow, deliberate movements to avoid inadvertent contamination.

Microbiology Waste Materials

Accumulated waste materials can pose a contamination hazard. A microbiology

laboratory can become inundated with old cultures unless a well-organized system for
disposal of is in place. Even a few people can produce so much contaminated material
that if teams do not take care of their own materials someone will spend at least a week
just cleaning up the place. All cultures must be sterilized at 121 oC using the autoclave
before disposal or cleaning of lab ware. To make disposal as efficient as possible, please
get rid of materials you no longer need as soon as possible.

b. Medium Preparation

Medium preparation methods are similar for broth and agar. Preparation of nutrient for
microbial growth is as below:

i. Calculate and weight the nutrient according to the calculation. Please refer to the
example below:
Example: (40g/L) mean 40 g of nutrient powder and dissolved in 1000ml of distilled
water.

If you required 2 petri dish of nutrient, amount of water and nutrient require are:

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= 2x25x40g/1000ml
=2g

ii. Dissolve amount of nutrient into the water and boil. Observe and record your
observation.
iii. Sterile the nutrient in autoclave at 121oC for 15 min.
iv. Prepared laminar flow for aseptic condition
v. Pour nutrient agar into the Petri dish and allow the nutrient to solidify under aseptic
condition and normal environment condition.
vi. Incubate your sample using incubator at 37oC for 24 hours.
vii. Observe and record your data.

** Samples are in form of agar, broth and slant agar.

Result and Discussion

- Examine all cultures for the appearance of growth, which is indicated by turbidity
in the broth culture and appearance of growth on the surface of the slant and
dish.
- Record pour observation in the chart below:

Status: Under aseptic condition

Nutrient broth Nutrient agar Nutrient agar
slant
Growth (+) 0r (-)
Pigmentation (+) or (-)
Distribution of growth

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