*TITLE: IDENTIFICATION OF EQUIPMENT AND MATERIALS IN THE MICROBIOLOGY LABORATORY

AIM: TO IDENTIFY THE EQUIPMENT AND MATERIALS IN MICROBIOLOGY LAB

MATERIALS: Different equipment and materials are provided for identification

THEORY: The microbiology laboratory deals with isolating, identifying and performing antimicrobial
susceptibility testing for microorganisms like bacteria, fungi, protozoa, viruses, etc. These procedure
needs both the capable human resources and also equipment to aid those individuals to perform above
mentioned activities. In other words, microbiology laboratory is a place that is set aside for the practical
study of microorganisms. Owing to their small size, most microorganisms require special handling with
special equipment not only to be able to study them properly, but also to prevent the laboratory
workers from dangers of the disease causing ones. This type of laboratory is equipped with specialized
tools and equipment to handle microorganisms safely, as well as sterile environments and containment
systems to prevent the spread of infection and infectious agents.

PROCEDURE: Students are to identify and familiarize themselves with the laboratory equipment and
materials

ACTIVITY:

- List 10 equipment and materials and state the function of each

- Draw at least 5 eqiupment and materials
*TITLE: PREPARATION OF CULTURE MEDIUM (LIQUID AND SOLID)

AIM: TO PREPARE LIQUID AND SOLID MEDIA

MATERIALS: Microscope, pond water, pipette, cover slip, clean glass slide, glass slide with mounted
specimen

THEORY: Culture media are mediums that provide essential nutrients and minerals to support the
growth of microorganisms in the laboratory. Microorganisms have varying nature, characteristics,
habitat, and even nutritional requirements, thus it is impossible to culture them with one type of culture
media. However, there are also microorganisms that can’t grow on a culture media at all in any
condition – these are called obligate parasites. Culturing microorganisms is essential for diagnosing
infectious diseases, obtaining antigens, developing serological assays for vaccines, genetic studies, and
identification of microbial species. Furthermore, it’s also essential for isolating pure cultures, storing
culture stock, studying biochemical reactions, testing microbial contamination, checking antimicrobial
agents and preservatives effect, testing viable count, and testing antibiotic sensitivity.

PROCEDURE:

- Weigh the amount of ingredients powder on weighing machine.

- Dissolve the ingredients in distilled water.
- Adjust PH of the medium if needed.
- Add agar and boiled it to dissolve.
- Pour the media into flask.
- Autoclave the media when ingredients fully dissolve.
- Sterilization is done in autoclave to prevent from contamination, at 121ºC for 15 min at 15lbs.
- After the autoclave place the media flask in laminar air flow.
- Sterilize the laminar air flow with 70% alcohol.
- A bit cools down the media and pours into sterile Petri-plates for solidification.
- Then sample is ready to spread(spreader) / streak
- (Inoculation loop) on the medium for identification or isolation of microbes.
- Sealed the Petri plates with paraffin, label them.
- Keep them inverted in incubator at 37ºC for 24hrs.
- Observe the result next day colonies formation is visible on the media.

ACTIVITY:

- List and explain types of culture media

*TITLE: ISOLATION OF MICROORGANISMS

AIM: TO PREPARE PURE CULTURE FROM MIXED CULTURE

MATERIALS: Sterile Petri dishes, L-shaped Bent glass, bunsen burner, Inoculation loop, rolled tissue,
marker pen, Micropipette, Mixed culture of isolated microbes, 10 mL solid NA media in a test tube, 10 mL
solid nutrient agar media in a Petri dish, 70% alcohol solution, 5 mL sterile distilled water in a test tube
THEORY: Microbial populations in nature consist of various species that live together. We can
separate the population into pure cultures in the laboratory. This pure culture only contains one type of
organism and is not mixed with others. Pure cultures are prepared to study the morphology and
biochemical properties of these microbes. The method used to obtain pure culture is by isolation. The
isolation technique determines the success of obtaining a pure culture that is separate from other
cultures. There are two ways of isolation to obtain pure culture, namely:
1. Streak plate method
In principle, this method is to scratch a suspension of material containing microbes on the surface of the
medium. The technique can use the 4 quadrant technique. After incubation, the scratch marks will grow
into separate colonies (Figure 1).

Figure 1. Four quadrant technique in the scratch method

2. Spread Method
The spread method consists of two techniques, i.e. spread plate and pour plate.
a. Spread plate is a spreading method by inoculating isolates on solid agar media in Petri dishes and
spreading using a sterile L-shaped glass spreader.
b. Pour plate is a spreading method by inoculating the isolate on solid agar media which melts at a
temperature of around 40°C-50°C, and then is poured into a sterile Petri dish.
The difference between these two is that the microbes that grow using the spread plate technique are
aerobic, whereas with the pour plate method, the growth can be aerobic (on the surface of the agar)
and facultative anaerobic/anaerobic (below the surface of the agar).
PROCEDURE:
Pure culture isolation technique using the four quadrant scratch method.
- Select a single colony in the mixed culture to isolate.
- Aseptically, take the colony using an inoculation loop and transfer it to NA media in a Petri dish
with the four-quadrant technique (Figure 1).
- The first step was carried out by dividing four parts of NA media in a Petri dish as shown using
markers (1, 2, 3, and 4).
- One colony is taken using an inoculation loop and scratched into area one.
- Next, the inoculation loop is burned using Bunsen and scratched from area one to two and
further scratches are made on area two without touching area one.
- This process is done successively until the scratches reach area four.
- Next, incubated in an incubator at 30°C for two days, then colony growth is observed.
- The indicator of the success of this process is that a single colony is obtained that is separate
from the others.
Pure culture isolation technique using the spread plate method.
- One colony is taken using an inoculation loop and placed in 5 mL of sterile distilled water and
homogenized using a vortex to obtain a culture suspension.
- Next, 100 μL of the culture suspension is taken using a micropipette, poured into NA medium in
a Petri dish and spread evenly using an L-shaped bent glass.
- Next, incubated in an incubator at 30°C for two days, then colony growth is observed.
- The indicator of the success of this process is that a single colony is obtained that is separate
from the others.
Pure culture isolation technique using the pour plate method.
- A total of 100 μL of culture suspension (from Step 2) was taken using a micropipette and poured
into liquid nutrient agar media in a test tube (temperature 40°C-50°C)
- Then homogenize by rotating the Petri dish to resemble the number eight.
- Incubate in an incubator at 30°C for two days, then observe the growth of the colony. The
indicator of the success of this process is that a single colony is obtained that is separate from
the others.

ACTIVITY:

- Differentiate between pure culture and mixed culture

- Explain the terms subculture and isolates
*TITLE: INCUBATION OF BACTERIA AEROBICALLY AND ANAEROBICALLY USING INCUBATOR
AND ANAEROBIC JAR
AIM: TO INCUBATE MICROORGANISMS USING INCUBATOR AND ANAEROBIC JAR
MATERIALS: An obligate aerobe (Pseudomonas spp), a facultative anaerobe (Streptococcus spp),
Inoculating loop, Bunsen burner, anaerobic jar, incubator, sterile nutrient agar plates
THEORY: Incubation conditions are crucial for the successful cultivation of microorganisms. By carefully
controlling the temperature, atmosphere, and duration of incubation, we can create an environment
that supports the growth of potential pathogens while suppressing the growth of contaminants,
ultimately leading to accurate and timely diagnoses. The atmospheric conditions during incubation can
have a significant impact on the growth of microorganisms, particularly those with specific oxygen
requirements (Aerobic: Normal atmospheric air; 21% oxygen, Anaerobic: Absence of oxygen). The
methods for Creating Specific Atmospheres includes (Aerobic: Incubate plates in a standard incubator
with free access to air; Anaerobic: Use anaerobic jars or chambers with chemical gas generators or
vacuum pumps to remove oxygen).

PROCEDURE:

- Inoculate the anaerobe by streaking with the aid of the inoculating loop on the surface of a
sterile nutrient agar plate
- Incubate anaerobically using anaerobic jar
- Inoculate the aerobe by streaking with the aid of the inoculating loop on the surface of a sterile
nutrient agar plate
Incubate aerobically using the incubator
- After incubation, examine the pattern of growth and observe under the microscope

ACTIVITY:

- Explain the terms anaerobes and aerobes

- Name 5 anaerobes and aerobes
- Draw the anaerobic jar and label its parts
- Explain the terms obligate and facultative as related to microorganisms

*TITLE: MAKING FILMS ON GLASS SLIDE

AIM: TO PREPARE SMEAR/FILM ON GLASS SLIDE

MATERIALS: clean glass slide, culture of a known organism, Bunsen burner, Inoculating loop

THEORY: A proper study of microorganisms requires techniques for preparing the microorganism(s)
before observing under the microscope. One of these techniques involve making a film. To make a film,
a smear of the organism to be observed is prepared and heat fixed. It is important for microbiologists to
master the art of making films because specimens to be observed under the microscope must be
prepared as a film on a glass slide prior to observation. Bacterial smear can be defined as a dehydrated
or dried preparation of a bacterial suspension (cells) on a clean glass slide. The source of bacteria for the
preparation or making of a smear can be from an agar slant, broth (liquid) culture and from a culture
plate.

PROCEDURE:

- Clean a plain microscope slide thoroughly using lens tissue.

- Label a microscope slide with a marker pen to record the culture being used, date
and initials; this is also a useful reminder of which side of the slide is being used.
- Flame a wire loop to ensure that no culture accidentally remains from a previous
operation.
- Transfer one or two loopfuls of tap water on to the centre of the slide.
- Flame loop and allow to cool.
- Using aseptic technique, transfer a very small part of a single colony from a plate or
slope of agar medium into the tap water.
- Make a suspension of the culture in the tap water on the slide and thoroughly but
gently spread it evenly over an oval area of up to 2 cm length.
- Flame the loop.
- Dry the suspension by warming gently over a Bunsen burner flame and then ‘fix’ it
by quickly passing it through the flame a few times.

ACTIVITY:

- Why did you emulsify the drop of water with the smear?
- Why did you heat fix?