Name: GALINATO, Jewel R.

Date of Experiment: May 10, 2021

Year and Section: BSN 1A

Experiment No 4 Inoculation Techniques

II. INTRODUCTION

I. LEARNING OBJECTIVES
At the end of the exercise, the students should be able to:
1. explain the rationale behind the use of aseptic culture technique;
2. practice the aseptic transfer of bacteria from one culture medium to another; and
3. demonstrate the different methods of inoculating microcrobes in agar plate and agar slant.

In the laboratory, microbes must be cultured in order to facilitate identification and to examine
their growth and metabolic characteristics. Microbes are inoculated or introduced into various forms of
culture media to keep them alive for study. Inoculation must be done through aseptic technique to
prevent contamination with unwanted microbes or contaminants. This technique usually involves
disinfecting the working areas, minimizing contact with contaminates, and using flames to eliminate
microbes which might enter vessels such as test tubes and petri dishes when they are opened.
There are three different inoculation methods used for isolation of bacteria: streak plate method,
spread plate method, and pour plate method. The spread plate method and the pour plate method are
quantitative techniques that allow determination of microorganism population in a sample. The streak
plate method is the most common isolation technique that uses sterile, heat-resistant, and non-corroding
nichrome wire attached to an insulated handle called inoculating loop (a wire bent into a loop) or
inoculating needle (a straight wire) in transferring bacteria in different culture media. For special
purposes, cultures may also be transferred with sterile cotton swabs, pipettes, glass rods, or syringes,
depending on the medium and technique.

III. MATERIALS
 Prepared cultured media:  Inoculating loop and needle
nutrient agar plate, nutrient agar  Inoculating needle
slant, nutrient agar deep, and  Screw cap or cotton plug
nutrient broths  Alcohol lamp
 Parafilm/adhesive tape
 Inoculum (sample from soil,
water, normal flora, food
sample)
 Pure bacterial culture:
Staphylococcus aureus and
Escherichia coli
 Incubator
IV. PROCEDURES
Streak plate method in agar plate
1. Flame-sterilize the inoculating loop until red hot. Allow it to cool for few seconds. Open and
flame-sterilize the neck of the test tube or lid of the petri dish containing pure culture and
insert the inoculating loop and pick up a loopful of the specimen. Flame-sterilize the opening
again and close immediately.
2. Hold the prepared sterile covered petri dish with your other hand. Partially open the lid of the
plate near the flame of the alcohol lamp.
3. Place a loopful of specimen on the surface of the agar medium away from you and streak the
culture back and forth without overlapping.
4. When the entire culture medium has been streaked, withdraw the inoculating loop and flame-
sterilize the entire cover of the plate and the loop.
5. Label and seal the petri dish using parafilm and place the plate in an inverted position in the
incubator at 37°C for 18 to 24 hours.
Streak zigzag method in agar slant
1. Take out a loopful of bacterial culture using a flamed-sterilized loop.
2. Hold the agar slant and remove the screw cap or cotton plug with your other hand. Do not
allow the cotton plug or screw cap of the test bacterial culture to touch unstrile surfaces.
3. Pass the mouth of the test tube through the alcohol lamp's flame. Quickly insert and inoculate
the agar surface by moving the inoculating loop from the bottom to the top of the agar slant in
zigzag or snake-like motion.
4. Flame-sterilize the opening of the tube and cover the agar slant.
5. Label the agar slant tube and place it in the test tube rack, then incubate it in upright position
at 37°C for 18 to 24 hours.
Suspend and shake method in inoculating nutrient broth
1. Follow steps 1 and 2 on how to inoculate an agat slant. Inoculate the medium from top to
bottom. Assume equal distribution of inoculums by suspending the inoculating loop and
shaking it.
2. Flame-sterilize the mouth of the test tube and the loop before and after inoculation.
3. Sterilize the screw cap; cover and label the broth tube; and place it in a test tube rack.
Incubate it in upright position at 37°C for 18 to 24 hours.
Stab method in agar deep/butt
1. Follow steps 1 and 2 on how to inoculate an agar slant using the inoculating needle.
 2. Transfer the inoculum into the agar deep or butt by dipping the inoculating needle into the
agar halfway and at the middle without touching the sides of the tube.
3. Flame-sterilize the mouth of test tubes and inoculating needle before and after the inoculation.
4. Sterilize the screw cap; cover and label the agar deep; and place it in the test tube rack.
Incubate it in upright position at 37°C for 18 to 24 hours.
V. ILLUSTRATIONS

A. Construct a schematic flow chart of the sterilization process of the inoculating loop and
needle with illustration.
B. Draw the steps in the inoculation of the following:
Streak plate method in agar plate

Streak zigzag method in agar slant

Nutrient broth
 Agar deep/butt

VI. RESULTS AND DISCUSSION

1. Explain importance of sterilizing the inoculating loop and needle before and after the inoculation
process.
In order to achieve the most accurate readings inside samples, inoculation loops must be sterilized
before and after each usage to reduce the possibility of infection as much as possible. If the
inoculating loop was not sterilized until being used to establish a new culture, cross contamination
is very possible. During an experiment, you want to reduce the amount of data collected, and
sterilization accomplishes this.
2. Outline the aseptic technique.
The use of procedures and techniques to prevent pathogen exposure is referred to as aseptic
technique. It requires adhering to the strictest regulations in order to minimize the risk of infection.
Healthcare practitioners use aseptic procedures in emergency rooms, hospitals, long-term care
centers, and a variety of other environments. The aseptic technique is made up of four main
components: barriers, patient equipment and preparation, environmental controls, and contact
guidelines. Each plays an important role in infection management during certain medical
procedures. The more humans there are, the more likely it is that infectious bacteria can
contaminate the environment. In the room, there are just one or two providers and the patient.
3. State the purpose of flame-sterilizing the mouth of the tube and the side of the Petri dish during
inoculation.
Carrying the loop into the blaze of the Bunsen burner kills all contaminating species and effectively
sterilizes everything. The loop must glow red-hot for several seconds. Enable the loop to cool
slightly after flaming before picking species from the inoculum culture (the culture that is to be
transferred.) Cool the loop when transferring a culture from a plate by rubbing it against the agar's
very edge. The red-hot loop will sizzle as soon as it is inserted into the culture after transmitting
from a broth. When the loop comes into contact with the broth culture, it can cool immediately, but
wait one to two seconds before removing the inoculum loop from the tube. (Aerosols can be
generated when the hot loop comes into contact with microorganism-containing media.) This causes
some of the broth and bacteria to temporarily simmer, producing a bacterial aerosol. These
airborne bacteria have the potential to invade the respiratory tract or other areas of the body. So, if
you immerse the heat sterilized loop in the broth culture, you can hear a hissing sound, indicating
that the loop has not been properly cooled). This ensures that all other microorganisms enter the
mouth of the vessel and contaminate the culture or medium. Passing the bottle's mouth into a flame
creates a convection current that exists away from the opening, which helps in contamination
avoidance. The hot section of the flame should be higher than the bright blue inner 'cone,' and the
vessel should be pushed across the flame rather than fixed in place.

VII. GUIDE QUESTIONS

1. Cite the uses of agar plate, agar slant, agar deep/butt, and broth culture media.
Agar plates are the standard solid support material for growing microorganisms. Microbial
growth media contains nutrients and an energy source to fuel the microbes as they grow, and
agar to keep the media in a semi-solid, gel-like state.
Agar slants can be used to culture bacterial cells for identification. However, when placed
on a nutrient agar slant, bacterial cells will divide quickly and within several hours will have
produced enough cells to examine microscopically. Agar slants are also useful in maintaining
bacterial cultures, more so than stacks of Petri dishes.
Agar deeps are used to grow bacteria that require less oxygen then is present on the
surface of the medium. They also aid in determining oxygen requirements and motile ty of
bacteria. Motile bacteria will grow/ move away from the point of inoculation.

2. Differentiate the streak plate method from the pour plate method.
The pour plate method is typically used to count the number of colony-forming
bacteria present in a liquid specimen. The pour plate method of counting bacteria is
more accurate than the streak plate method, but it yields a lower count on average since
heat sensitive microorganisms may die when they come into contact with thick, molten
agar medium. Spread plate technique, on the other hand, is a way of isolating and
enumerating microorganisms in a mixed culture and spreading them uniformly. The
technique simplifies the quantification of bacteria in a solution.
3. Indicate the correct way of inoculating the following media.
Plate media
A. For sensitivity testing
For sensitivity testing the depth of the agar is usually recommended to be 4mm in the centre of the plate
(approximately 25ml in a 90mm plate). Variation in depth will affect the zone sizes – if the agar is too
thin, larger zones will appear since the volume is decreased, and the effective antibiotic concentration
increased. If the agar is too thick, smaller zones will appear since the effective antibiotic concentration
has been decreased. If the agar is intentionally thin, then small modifications to other factors will have a
disproportionate effect.
B. For isolation of colonies
For isolation of colonies to obtain an isolate, flame-sterilize an inoculating loop or needle or use a sterile
disposable toothpick or stick. Exercising aseptic technique, touch the colony so that some material
adheres to the end of the instrument. Aseptically transfer the material to a fresh agar plate and streak it out
using the dilution streak method. Most colonies have a soft texture and can be sampled readily. Use a
loop if you have plenty of clearance. Then you can use the same loop to do the dilution streaking. Use a
needle if a loop is too big to enable you to sample from just that one colony.

VIII. CONCLUSIONS
Microorganisms can be found on all inanimate objects, making them common
sources of infection in the laboratory. The ability of a scientist to sterilize work surfaces
and equipment, as well as avoid contamination of sterile instruments and solutions with
non-sterile surfaces, is critical to the effectiveness of an experiment. Usually, this
method entails disinfecting work areas, reducing contact with pathogens, and using
flames to destroy bacteria that can enter vessels such as test tubes and petri dishes
when they are opened. The key problem when doing microbiology challenge research is
deciding on and validating an inoculation process. To effectively detect the bacterial
content of specimens, individual colonies must be grown by using a good technique to
inoculate the specimen on culture media. All five techniques use aseptic approach, or
techniques that keep raw materials sterile. (1) streak plating bacterial cultures to
separate single colonies, (2) pour plating and (3) scatter plating to enumerate viable
bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5)
replica plating to move cells from one plate to another in an identical spatial pattern are
among the procedures mentioned. These experiments may be carried out at a laboratory
bench if they contain non-pathogenic microorganism strains.

ONLINE RESOURCES

Bacterial Sampling. Retrieved from virtuallabs.nmsu.edu on March 10, 2021

Media Preparation. Retrieved from www.ncbionetwork.org/educational-

resources/elearning/media-preparation on March 10, 2021.