Edzel R.

Bajado BS BIOLOGY – II
BIOTURORIAL I January 12, 2024
F (11:00-12:00 PM)
ACTIVITY 1
Mix Culture

PROCEDURE

• Prepare a serial dilution of your mixed sample.

• Transfer 1 ml of each dilution into a separate Petri dish.
• Pour molten agar into the Petri dishes.
• Rotate the plates to ensure mixing of the sample and agar.
• Incubate the plates
 ACTIVITY 2
Streak Plate

PROCEDURE
• Sterilize the wire loop.
• Cool the loop by touching it on the edge of the sterile agar plate.
• Dip the loop into the broth culture containing the mixture of bacteria.
• Lift the lid of the plate just enough to insert the loop. Drag the loop over the surface
of the top one-third of the plate back and forth in a "zig-zag" formation.
• The loop has picked up thousands of bacteria which are spread out over the surface
of the agar.
• Sterilize the loop in the flame.
• Turn the plate 90 degrees and drag the loop through the area you have just streaked
two to three times and continue to drag the loop in a "zig-zag" formation in the
remaining half of the plate without touching that area again.
• Sterilize the loop in the flame.
• Turn the plate 90 degrees. Repeat the procedure. Drag the loop two to three times
through the area you just streaked, and fill in the remaining area of the plate (zig-zag
formation), being very careful not to touch any of the areas you previously streaked.
• Incubate the plate for 24 hours. If you streaked correctly, you will see isolated colonies
in the third sector. The heaviest growth will be in the first sector. There will be less
growth and some isolated colonies in the second sector. The third area should have
the least growth with isolated colonies.
 ACTIVITY 3
Slant Isolation

PROCEDURE
• Sterilize the wire loop.
• Cool the loop by touching it on the edge of the sterile agar plate.
• Dip the loop into the broth culture containing the mixture of bacteria.
• Lift the lid of the plate just enough to insert the loop. Drag the loop over the surface of the top onethird of
the tube back and forth in a "zig-zag" formation.
• The loop has picked up thousands of bacteria which are spread out over the surface of the agar.
• Sterilize the loop in the flame.
• Turn the tube 90 degrees and drag the loop through the area you have just streaked two to three times and
continue to drag the loop in a "zig-zag" formation in the remaining half of the plate without touching that
area again.
• Sterilize the loop in the flame.
• Turn the tube 90 degrees. Repeat the procedure. Drag the loop two to three times through the area you just
streaked, and fill in the remaining area of the plate (zig-zag formation), being very careful not to touch any
of the areas you previously streaked.
Incubate the tube for 24 hours. If you streaked correctly, you will see isolated colonies in the third sector.
The heaviest growth will be in the first sector. There will be less growth and some isolated colonies in the
second sector. The third area should have the least growth with isolated colonies.
 ACTIVITY 4
Staining (simple staining, gram-staining, negative)

SIMPLE STAINING GRAM STAINING NEGATIVE STAINING

Negative Staining
• Take two slides
• Place a drop of acidic dye, nigrosin, at one end of your slide.
• Place your specimen into the dye and swirl it in the stain.
• Using the other slide, hold it at a 45º angle and feather the mixture across the slide by
pushing the dye.
• Air dry but do NOT heat fix. This will keep the morphology of the bacteria intact.
• Observe your negative slide
Gram Staining
• Stain with primary stain (crystal violet) for 1 min.
• Apply mordent (Iodine) for 1 min.
• Rinse with decolorizer like ethyl alcohol (15 - 30 sec)
• Stain with counter stain (safranin) for 30 sec.
• Rinse between each of the above steps. Blot dry and observe.
Simple Staining
• Make an air dried, heat fixed smear of your bacteria sample.
• Stain with a basic dye (methylene blue) for 3 minutes.
• Rinse, blot dry, and observe
 ACTIVITY 5
Pour Plate

PROCEDURE
• Ensure all instruments, flasks, and media required for the procedure are sterilised.
• Clean your workspace using a disinfectant to minimise potential contamination.
• Set up a Bunsen burner in your workspace, ensuring it is done so safely.
• Wash your hands with an antiseptic solution before handling any microbial samples.
• Label the Petri dish with relevant information including your name, date, the media used,
and the culture being inoculated.
• Sample Preparation: If the sample is semisolid or solid, suspend it in sterile water or broth
to create a liquid solution. If the sample is already liquid, prepare serial dilutions to reduce
the microbial colony load to a range of 20-300 CFU/ml. Dilutions can be prepared up to
10.
• For inoculation, open the Petri dish lids and pour 1 ml of the diluted sample. Take the
molten agar, heat it slightly and pour about 15-18 ml onto the sample. Ensure the agar is
not too hot or too cold. Close the dish lid and swirl gently.
• Another method for inoculation is to mix the diluted sample in the agar medium, mix it
gently, and then pour it into the Petri dish. This method is less common.
• Allow the plate to solidify. Invert the plate and incubate it at an optimal temperature
(usually 37℃) for 24-48 hours.
 ACTIVITY 6
Spread Plate

Procedure
• Arrange all the requirements, put on the PPE, sterilize the work surface, and
allow all the samples and media to come to room temperature if were
refrigerated.
• Sample preparation
• Liquid samples must be serially diluted to reduce the microbial load to the
range of 20 – 300 CFU/mL. (If the sample is assumed to be sterile or the expected
microbial load is very low, we can escape the dilution. The prior pilot test may give
an exact value. You can prepare serial dilution up to 10 -10 and use different
dilutions.)
• If the sample is solid or semisolid, dissolve it with a suitable solvent to prepare
its suspension. The suspension then should be serially diluted to reduce the
microbial load at the desirable range. (Generally, 1 gm sample is mixed with 9
ml of solvent to get the concentration of 10-1 gm/mL.)
• Arrange the spreader. The glass rod must be sterilized. For this dip the rod in
70% ethanol solution and flame it over a Bunsen burner. Let the rod cool. (You
can check if the rod is cold enough or not by touching a corner of solidified media.
If you heard a sizzling sound or if the media melts, then cool the rod further.)
Likewise, the beads can be put in a bottle or beaker and then autoclaved to
sterilize them.
• Label the plate at its bottom edge with the dilution factor, date, name, sample
ID, and other required information.
 ACTIVITY 7
Endophytic Fungi

Procedure
• Collection of plant material
• In choosing a plant species, study its geographic location, collect the parts of the plant
and the number of samples needed.
• Surface disinfection and Endophytic Isolation
• The next step is the chemical sterilization. The tissue is then subjected to ethanol
immersion, the primary sterilizing agent (sodium hypochlorite and mercury chloride),
and distilled water that has been autoclaved before being cleaned.
• Next is the physical sterilization which we can use sonication or ultraviolet light.
• To cultivate the endophytic fundi, the plant tissues will be divided into smaller sections
and placed in growth media (such as Hagem minimum medium or malt extract agar).
Antibiotics will also be used to suppress endophytic bacteria in order to study the
diversity of the endophytic fungi.
• Incubate the cultured plates at a temperature of 25–28 °C for 3–20 days, which can be
extended up to 6 weeks if necessary.
• The next step is isolation, which involves collecting the hyphae from the fungus colonies'
borders and replanting in new culture media. You should keep doing this until the colony
is uniform. Then monosporic or hypha tip purification must be performed.
 ACTIVITY 8
Purification endophytic fungi

Procedure
• Leaf samples were obtained and cut into small, square-shaped discs.
• The discs were surface sterilized by submerging in 10% ethanol (CH₃CH₂OH) for 3
minutes, then exposing to 3% sodium hypochlorite (NaOCl) for 10 seconds, soaking again
with 10% CH₃CH₂OH for 3 minutes, and lastly, rinsing them twice with sterile distilled
water (Supaphon et al., 2014).
• Finally, the discs were dried by blotting them with sterile tissue paper, then inoculated in
corn meal agar or CMA culture medium (2.55 g per 150 mL of CMA; 0.0255 g of
chloramphenicol antibiotic) at random areas, and incubated for 7 days.
• For plates with successful growth of endophytic fungi, mycelial discs were cut and
inoculated in another sterile CMA culture medium.
• For sporulating fungi, only the spores were taken using a dissecting needle. The media
with inoculated fungi were then incubated.
 ACTIVITY 9
Antagonistic Activity

PROCEDURE
 Fungal Strains
Select the fungus that you want to co-culture. Choose the antagonist fungus and the test
fungus.
Indicate where the fungal strains came from, whether it was from environmental
samples or culture collections. Specify the preservation method (such as
cryopreservation) used for the fungal strains.
 Culture Condition
- Describe the growth medium's composition that was utilized to cultivate the
fungi strains. Add the liquid media, agar plates, and any additives or selective
agents that were utilized.
- Explain the production of the spore or mycelial inoculum and the inoculation
procedure for each fungal strain.
- Describe the incubation conditions for the growth of the fungus, including the
temperature, light conditions, and length of time.
 Co-culture
- Describe the co-culture technique used, including any physical separation or
direct interaction.
- To compare the outcomes, include the proper controls, such as mono-cultures
of the test and antagonist fungi.
 ACTIVITY 10
Anti-bacterial activity of crude extract

PROCEDURE
• First, the exhausted culture medium was vacuum-filtered by gradually reducing the filter
pore size (From 10 to 0.22-μm).
• The filtrate was then concentrated to 40ml by vacuum-evaporation (40°C, 20mbar) and
extracted thrice with EtOAc (1:1 w/w) in a separation funnel.
• The organic phases followed the same procedure as in section Mycelial Organic Crude
• Extract to finally obtain extracellular crude extracts (ECEs)