Microbiology Lab: Enumeration, Growth, and Microbial Influences Study

This microbiology lab report details several techniques used to enumerate microorganisms, including plate count, direct microscopic count using a counting chamber, and turbidity measurement. The experiment investigates the impact of various environmental factors on microbial growth, such as temperature, pH, osmotic pressure, and the presence of metals. The methods section outlines the procedures for serial dilution, pour plating, spread plating, and swab sampling. The objectives include comparing enumeration methods, determining the effect of temperature and pH on different microbial species, and assessing the responses of bacteria and fungi to solute concentrations and metals. The results section, which is not fully provided in the given text, would present the findings from these experiments, leading to conclusions about the influence of these factors on microbial growth. The report also references the growth characteristics of mesophiles, thermophiles, hyperthermophiles, psychrophiles, acidophiles, neutrophiles, and alkaliphiles.

INTRODUCTION
There are various instances in the field of microbiology when it is indispensable to
either determine or estimate the count of microorganisms in a given sample. For
example, food microbiology, dairy microbiology and water microbiology are
dependent on the quantification of level of microorganisms in the products from
these industries. Quite a number of methods are being used for the enumeration of
microorganisms either directly or indirectly. Some of these methods which are being
used in this exercise are plate count technique, direct microscopic count using a
counting chamber and turbidity measurement.
Standard Plate Count (VIABLE COUNT)
Standard plate count or viable count method is technique in which a cell or a clump
of cell divides to form a colony and hence they are known as colony-forming units or
CFU. The plate count technique usually involves serial dilution of the sample, and
plating of the dilution aliquots onto a suitable agar growth medium to detect colonies.
The number of colonies reflects the number of organism contained in the sample that
was capable of growing under the specific condition of culture and incubation. The
number of viable bacteria in a sample is expressed as the number of CFU/ml o the
sample.
Direct Microscopic Count (TOTAL CELL COUNT)
Direct Microscopic Count using counting chambers (Haemocytometer), consisting of
a ruled slide and a coverslip, is being used as a direct method to count the number
of bacterial cells in a liquid sample. In this technique, the number of cells in a given
volume of sample is counted directly under the microscope using Haemocytometer.
The average of number of cells is calculated and then the number of cells ml-1 of
sample can be determined.
Turbidity Measurement
The concentration of cells in a liquid medium can be estimated by an efficient and
simple technique that measures the turbidity (cloudiness) of a culture using a
spectrophotometer. A spectrophotometer is a device that measures the amount of
light absorbed at various wavelengths by the sample in terms of photons after it
passes through the sample. Spectrophotometer consists of a light source, a filter, the
sample tube, and a photocell. The determination of concentration of cells using
turbidity method is based on the measurement of optical density (O.D) of the sample
and then plotting this O.D in a standard curve plot of know concentration of culture in
terms of cells/ml. Before the measurements of turbidity can be made, the
spectrophotometer must be set to 100% transmittance (0% absorbance). This is
done using an uninoculated medium.

The growth rate of microorganisms is affected by many external factors and thus
these factors play important role in controlling the growth of microorganisms in many
microbiological industries (Kaiser, 2019).
Effect of Temperature
Bacteria have a minimum, optimum, and maximum growth temperatures. Mesophiles
are microorganisms that grow at moderate temperatures. Their optimum temperature
of growth is between 25°C to 40°C. Thermophiles are the heat-loving
microorganisms. Their optimum growth temperature is between 50°C to 60°C.
Hyperthermophiles are bacteria whose optimum growth temperature is between
70°C to 110°C. Psychrophiles are cold-lovers. Their optimum growth temperature is
between 2°C and 20°C.
Here, the growth characteristics of different species of microorganisms at different
temperatures will be determined.
Effect of pH
The pH of the surroundings influences the growth of microorganisms. Acidophiles
grow at their optimum at a pH below 5.5. Neutrophiles grow at a range of pH from o5
to 8. Alkaliphiles grow best at a pH above 8.5.
Effect of Osmotic Pressure
Osmotic Pressure is the resultant of Osmosis which is the diffusion of water across a
semi permeable membrane from an area of higher water concentration to an area of
lower water concentration. The addition of sugars and salts alters the osmotic
pressure making it hard for the microorganisms to grow. Thus the group of
microorganisms that can bear with the high osmotic pressure is known as
osmophilic. Increasing osmotic pressure to inhibit the growth of micro organisms has
been applied in food industries.
Effect of Metals
There are certain metals which are critical for the functioning of the microorganism at
a very low concentration and hence they are known as trace elements. However,
these metals can exert inhibitory action against the micro organisms at their higher
concentrations.
OBJECTIVES:
 To enumerate microorganisms in a sample comparing plate count, direct
microscopic and turbidity measurement methods.
 To determine the effect of temperature on different species of
microorganisms.
 To compare the effect of pH on fungal and bacterial growth.

 To determine the responses of bacteria and fungi to solute concentrations of
salts and sugars.
 To demonstrate the effect of metals on bacteria.
METHODS:
Procedure of serial dilution
1. 1 ml of culture of Saccharomyces cerevisae was pipette out and put in the 9
ml of peptone water labelled as 10-1. 10-fold serially diluted samples were
prepared ranging from 10-1 to 10-4.
Procedure of pour plating
1. One bottle of sterile molten plate count agar was collected from the water bath
maintained at 50°.
2. 1 ml aliquot from each of the dilution was transferred in sterile petri plates.
3. 15-20 ml of molten agar was poured in each of the plate and mixed carefully.
4. The plates were allowed to cool and incubated overnight at 37°C
Procedure of spread plating
1. One bottle of sterile molten plate count agar was collected from the water bath
maintained at 50°.
2. 15-20 ml of molten agar was poured in each of the plate, allowed to cool and
solidify completely.
3. 0.1 ml aliquot from each dilution was spreaded using glass spreader. The
plates were incubated overnight at 37°C
Procedure of swab sampling
1. Area to be examined was selected
2. A swab was removed from the swab container and immersed in the peptone
water. Excess fluid was pressed out on the neck of the bottle.
3. Swab was rubbed up and down, at right angle and at 45 ° angle over whole of
the exposed area of the template.
4. Swab was put back into the peptone water and stick was broken down such
that 2-4 cm off handle remain attached to the head.
5. Bottle was closed and shaked vigorously for 5-10 times.

6. 0.1 ml aliquot was used for spread plating and 1 ml of aliquot was used for
pour plating.
Procedure of enumeration of cells through counting chambers
1. Using a sterile pipette, 1-2 drops of the culture was put on the platform of
counting chambers and carefully covered with clean and dry over slip
ensuring no air bubbles.
2. Cells were counted.
Procedure of enumeration of cells through Turbidity measurement
1. 1 ml aliquot of serially diluted samples was taken and optical density was
measured at 650nm using uninoculated Nutrient Broth as blank in a
spectrophotometer.
RESULTS:
CONCLUSION:
Kaiser, G. (2017, April). Factors that Influence Bacterial Growth Retrieved from
https://bio.libretexts.org/Bookshelves/Microbiology/Book
%3A_Microbiology_(Kaiser)/
Unit_7%3A_Microbial_Genetics_and_Microbial_Metabolism/
17%3A_Bacterial_Growth_and_Energy_Production/
17.2%3A_Factors_that_Influence_Bacterial_Growth