BIMT 414 - Lec 2.

BIMT 414: MICROBIOLOGY –
Lecture 2
Mr. Kanchana Dassanayake (M. Sc in Microbiology)
27dassanayake@gmail.com
Kanchana Dassanayake(M.Sc in Microbiology)

https://www.youtube.com/watch?v=ZqlJSEu0Eo4&t=21s

https://www.youtube.com/watch?v=N1780tJTk90&t=1s

Dark-Field Microscope
• Bright Object, Dark Background
• The dark-field microscope produces
detailed images of living, unstained cells
and organisms by simply changing the
way in which they are illuminated.
• A hollow cone of light is focused on the
specimen in such a way that unreflected
and unrefracted rays do not enter the
objective.
• Only light that has been reflected or
refracted by the specimen forms an
image.
Use –
• To reveal considerable internal structure in larger eukaryotic
microorganisms.
• used to identify certain bacteria such as the thin and distinctively
shaped Treponema pallidum, the causative agent of syphilis.

Phase-Contrast Microscope
• A phase-contrast microscope converts slight
differences in refractive index and cell density
into easily detected variations in light intensity.
Usage –
• Studying microbial motility
• Determining the shape of living cells
• Detecting bacterial structures such as endospores
and inclusions

Fluorescence Microscopes
• The fluorescence microscope excites a specimen with a specific
wavelength of light (UV, violet, or blue light) and forms an image with
the fluorescent light emitted by the object.
• The most commonly used fluorescence microscopy is epifluorescence
microscopy.
• Specimen stained with special dye molecules called fluorochromes/
fluorescent dyes (Acridine orange, acridine yellow, fluorescein, eosin).

Microbial Growth
• Microbial growth = increase in number of cells, not cell size
The Requirements for Growth
• Physical requirements
• Temperature
• pH
• Osmotic pressure
• Chemical requirements
• Carbon
• Nitrogen, sulfur, and phosphorous
• Trace elements
• Oxygen
• Organic growth factor
• The survival and continued growth of microorganisms depend on an
adequate supply of nutrients and a favorable growth environment.
• A solution that containing these nutrients is a culture medium.
• Basically, all culture media are liquid, semisolid, or solid.
• Liquid medium lacks a solidifying agent and is called a broth medium.
• A broth medium supplemented with a solidifying agent called agar

results in a solid (1.5-2.0% agar) or semisolid medium depending on
the percentage of agar included.
- Semi-solid culture medium - Contains 0.5-0.75% agar (less than 1%). used
to demonstrate bacterial motility. Example: Mannitol mobility medium.

Aseptic Technique
• Sterility is the hallmark of successful work in the microbiology

laboratory, and sterilization is the process of rendering a medium or
material free of all forms of life.
• To achieve sterility, it is mandatory that you use sterile equipment and

employ aseptic techniques when handling bacterial cultures

https://www.youtube.com/watch?v=krZl4KKzYF8&t=49s

Colony-forming units (CFU)
• Desired CFU - 30 – 300 CFU/mL
• CFU < 30 – TFC (Too few to count)
• CFU > 30 – TMC – (Too more to count)
Count the colonies and calculate CFU/mL by using the formula:

• For example, suppose the plate of the 10-4 dilution yielded a
count of 27 colonies. Then, the number of bacteria in 1 ml of
the original sample can be calculated as follows:
• Bacteria/ml = (27) x (104 ) x 10 = 2.7 × 106

• In nature, microbial populations do not segregate themselves by
species but exist with a mixture of many other cell types.
• In the laboratory, these populations can be separated into pure

cultures.
• These cultures contain only one type of organism and are suitable for
the study of their cultural, morphological, and biochemical
properties.

Streak-plate method - for Isolation of Pure
Cultures
• A rapid qualitative isolation method.
• Involves spreading a loopful of culture over the surface of an agar
plate.
• The four-way, or quadrant, streak is the most common one.

A. Place a loopful of culture on the agar surface in Area 1. Flame the
loop, cool it by touching an unused part of the agar surface close to
the periphery of the plate, and then drag it rapidly several times
across the surface of Area 1.
B. Reflame and cool the loop, and turn the Petri dish 90°. Then touch
the loop to a corner of the culture in Area 1 and drag it several
times across the agar in Area 2. The loop should never enter Area 1
again.
C. Reflame and cool the loop and again turn the dish 90°. Streak Area
3 in the same manner as Area 2.

D. Without reflaming the loop, again turn the dish 90° and then
drag the culture from a corner of Area 3 across Area 4, using a
wider streak. Don’t let the loop touch any of the previously
streaked areas.
E. The loop was flamed and set aside. The agar plate was covered
and incubated at 370 C for 24 hours.
• The flaming of the loop at the points indicated is to dilute the culture
so that fewer organisms are streaked in each area, resulting in the
final desired separation.

2. CONTINUOUS STREAK TECHNIQUE
1. The agar plate was marked into two half circles(or can use the all
surface area of the plate).
2. The loop was flamed and one colony was removed from the mixed
culture and one of the half circle was streaked in a continuous back
and forth line.
3. The agar plate was rotated to 180 degree and the same was done in
these on half circle without flaming the loop.
4. The loop was flamed and set aside. The agar plate was covered and
incubated at 370 C for 24 hours.

THREE SECTOR STREAK (T-STREAK)

https://www.youtube.com/watch?v=_1KP9zOtjXk&t=1s

Serial dilution

Sample preparation:
• If the sample is in liquid form, serially dilute it to make the microbial
load to the range of 30 – 300 CFU/mL. (Prior pilot test may give exact
value. You can prepare serial dilution up to 10-10 and use different
dilutions.)
• If the sample is in solid or semisolid form, dissolve it in sterile
distilled water, saline(0.85% NaCl) or sterile broth, or any other
solvent. (Generally, 1 gm sample is mixed with 9 ml of solvent to get
the concentration of 10-1 gm/mL.)

Spread-plate technique
Steps -
1. Pipette out 0.1 ml from the appropriate desired dilution series onto
the center of the surface of an agar plate.
2. Dip the L-shaped glass spreader (hockey stick) into ethanol. Ethanol
is used to sterilize the glass spreader.
3. Flame the glass spreader over a bunsen burner.
4. Spread the sample evenly over the surface of the agar using a cool
alcohol-flamed glass rod spreader, carefully rotating the Petri dish
underneath at an angle of 45 degree at the same time.
5. Incubate the plate at 37°C for 24-48 hours.

Pour-plate technique
Steps -
1. Dispense 1 ml of diluted sample in the center of the Petri plate
using a sterile micropipette or calibrated pipette.
2. Open the lid of the bottle and flame its mouth. Pour about 15 mL
(1/3 of the petri plate’s height) of sterilized molten media at the
appropriate temperature (40 – 450C ) above the sample.
3. Close the lid of the plate then mix the sample in the media properly
by gently swirling the plate. The plate is generally swirled in an “S”
or “8” shape.
4. Incubate the plate in an inverted position for 24 hours at 37°C.
https://www.youtube.com/watch?v=0X39eeZbS98
https://www.youtube.com/watch?v=0X39eeZbS98

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BIMT 414: MICROBIOLOGY –

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

• A broth medium supplemented with a solidifying agent called agar

Kanchana Dassanayake(M.Sc in Microbiology)

• Sterility is the hallmark of successful work in the microbiology

• To achieve sterility, it is mandatory that you use sterile equipment and

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Count the colonies and calculate CFU/mL by using the formula:

Kanchana Dassanayake(M.Sc in Microbiology)

• Bacteria/ml = (27) x (104 ) x 10 = 2.7 × 106

Kanchana Dassanayake(M.Sc in Microbiology)

• In the laboratory, these populations can be separated into pure

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

Kanchana Dassanayake(M.Sc in Microbiology)

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