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ERT 107- Microbiology for Bioprocess Engineering
Laboratory module
EXPERIMENT 1
BASIC TECHNIQUES IN MICROBIOLOGY
1.0 OBJECTIVES
1.1 To familiarize the techniques of medium preparation
1.2 To familiarize the techniques of serial dilution and spread plate method.
2.0 COURSE OUTCOME
CO1: Ability to acquire and apply knowledge of microbiology concepts and
techniques in bioprocess
3.0 INTRODUCTION
Bacteriological media come an a wide range of types. Nutrient Agar is a complex
medium because it contains ingredients with contain unknown amounts or types of
nutrients. Nutrient Agar contains Beef Extract (0.3%), Peptone (0.5%) and Agar
(1.5%) in water.
Dilution plate and spread plate technique the method could be used to isolate pure
culture. These methods are biased in favor of rapidly growing and sporulating
organisms, and consequently most of the fungi identified by these techniques are
Fungi Imperfecti (Pencillium and Aspergillus). In these methods, samples will be
diluted several times prior to spreading on the agar plate. Then the diluted sample
is placed on centered of an agar plate and spread evenly over on the surface with
the sterile-bent glass rod or known as “hockey” stick. After incubation with
appropriate temperature, the fungus cell will be developed into isolated colonies.
The isolated colonies are picked up and streaked on the fresh medium to ensure
its purity.
The diversity of bacteria present in our environment and on and in our bodies is
incredible. Bacteria usually exist in mixed populations. It is only in very rare
situations that bacteria occur as a single species. However, to be able to study the
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cultural, morphological, and physiological characteristics of an individual species,
it is essential, that the organism be separated from the other species that are
normally found in its habitat.
Several different methods for getting a pure culture from a mixed culture are
available. One of the techniques is Streak plate. The purpose of s streak plate is to
isolate individual bacterial cells (colony-forming units) on a nutrient medium.
Two major steps are involved in obtaining pure cultures from a mixed population:
1. First,
the
mixture
must
be
diluted
until
the
various
individual
microorganisms become separated far enough apart on an agar surface
that after incubation they form visible colonies isolated from the colonies of
other microorganisms.
2. Then, an isolated colony can be aseptically "picked off" the isolation plate
and transferred to new sterile medium. After incubation, all organisms in
the new culture will be descendants of the same organism, that is, a pure
culture.
4.0
MATERIALS AND EQUIPMENTS
4.1
Micro pipette (10µL, 100µL, 1000µL)
4.2
Inoculation Loop.
4.3
Bunsen burner.
4.4
Pure Cultures of E.coli, Bacillus sp, Saccharomyces cerevisiae
4.5
Nutrient agar plates (NA)
4.6
Potatoes dextrose agar plates (PDA)
4.7
Sterilized distilled water
4.8
Test tubes
4.9
70% ethanol
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5.0 PROCEDURES
EXPERIMENT 1A: MEDIUM PREPARATION
5.1 Preparation of Nutrient agar (NA)
Nutrient agar and broth are available commercially in powdered (free-flowing,
homogeneous) form.
5.1.1 Dissolve the dehydrated medium in the appropriate volume of distilled
water i.e., 23 g dehydrated nutrient agar (see the manufacturer
instruction) in 1000 ml distilled water.
5.1.2 Heat with frequent agitation and boil for 1 minute to completely
dissolve the powder
5.1.3 Sterilized the medium by autoclaving (121°C for 15 min)
5.1.4 Dispense the medium in to tubes or plates. Left the agar medium to
solidify and store.
5.2 Preparation of Potato dextrose agar (PDA)
Potato dextrose agar are available commercially in powdered (free-flowing,
homogeneous) form.
5.2.1 Dissolve the dehydrated medium in the appropriate volume of distilled
water i.e., 23 g dehydrated nutrient agar (see the manufacturer
instruction) in 1000 ml distilled water.
5.2.2 Heat with frequent agitation and boil for 1 minute to completely
dissolve the powder
5.2.3 Sterilized the medium by autoclaving (121°C for 15 min)
5.2.4 Dispense the medium in to tubes or plates. Left the agar medium to
solidify and store
.
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EXPERIMENTS 1B:
5.3 CULTURE ISOLATION THROUGH DILUTION STREAK PLATE
TECHNIQUE
5.3.1
Prepare your laminar chamber by disinfecting its surface with the
disinfectant that is available in the laboratory. Use cotton to scrub it
clean.
5.3.2
Label the bottom surface of a sterile petri dish containing agar
media with your full name, group and date. You have to submit your
Petri dish.
5.3.3
Heat the loop and wire to red-hot.
5.3.4
Remove the cover of a petri dish containing culture of microbe
slowly. Do not place the cover down on the table.
5.3.5
After allowing the loop to cool, take out a loopful of microorganisms
carefully.
5.3.6
Return the cover to the culture dish.
5.3.7
Aseptically, transfer the microorganisms on a corner of the agar
medium plate and streak the plate slowly (Figure 1). Care should be
taken not to gouge into the medium with the loop.
 Flame the loop until red-hot and cool it down for a while. Then,
make 5 to 6 streaks from area 1 through area 2
 Flame the loop again until red-hot and then let it cool for a
while
 Make another 5 to 6 streaks from area 2 through area 3
 Flame the loop again and make another 5 to 6 streaks from
area 3 through area 4, using the remaining of the plate
surface.
 Alternative streak patterns are shown in Figure 2.
5.3.8
Flame the loop before placing it down.
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Incubate the inoculated agar medium plate in an inverted position at
37°C for 24 to 48 hours. By incubating the plates upside down, the
problem of moisture on the cover is minimized.
Figure 1: Quadrant streak
Figure 2: Alternative streak patterns
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EXPERIMENT 1C:
5.4 SERIAL DILUTION AND SPREAD PLATE TECHNIQUE
5.4.1
Using aseptic techniques, add a loopful of yeast culture to the bottle of
10 ml sterilized distilled water. Mix by rolling the tube between your
palms.
5.4.2
Transfer 1 ml of the suspended culture from the first bottle into 9 ml of
sterilized distilled water in the second bottle. Mix the second tube by
rolling the tube between your palms.
5.4.3
Repeat the step 5.4.2 to the third bottle.
5.4.4
Pipette 1 ml of the suspended culture from the third bottle into prepared
PDA agar plate.
5.4.5
Spread evenly the suspended culture on the surface of agar plate and
make sure the culture completely covered the agar surface.
5.4.6
Invert the plate and incubate at 37°C for 72 hours.
1 loop
9ml sterilized
distilled
water each
1 ml to agar
plate
Complex colonial morphologies
Figure 3: The dilution and spread plate technique for isolation of pure culture.
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RESULTS AND DISCUSSIONS
You are required to report the observations made, draw and label
appropriately and to discuss the difficulties, if any, faced during your work.
You have to submit your own plated petri dish with pure culture.
7.0 REFERENCES
Ahmad-Anas, N.G. Studies on the production of glucose oxidase from
Aspergillus terreus UniMAP AA-1 (Master of Science Dissertation, Universiti
Malaysia Perlis, 2011).
Bauman, R. 2007. Microbiology with diseases by taxonomy 2nd Edition.
Pearson Education, Prentice Hall.
Messley, K.E. & Norrell, S.A. 2003. Principles and Application: Microbiology
Laboratory Manual. Prentice Hall, Upper Saddle River.
Zulkali, M.M.D. 2004. MICET Experiment Manual 2004. Universiti Sains
Malaysia, Pulau Pinang.
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EXPERIMENT 2
PREPARATION OF SMEARS AND STAINING OF
SPECIMENTS
1.0 OBJECTIVES
1.1
To prepare wet mount and bacterial smears for microscopic study.
1.2
To stain bacterial smears, observe cell morphology and typical cell
arrangements.
1.3
To differentiate the structure, morphology and typical characteristics of
bacteria, yeast and fungi.
1.4
To understand the basic principles underlying staining procedures.
1.5
To differentiate between gram-positive and gram-negative bacteria.
2.0 COURSE OUTCOME
CO1: Ability to acquire and apply knowledge of microbiology concepts and
techniques in bioprocess
3.0 INTRODUCTION
A bacterial smear is a dried preparation of bacterial cells on a glass slide. In a
bacterial smear that has been properly processed;
(i)
the bacterial are evenly spread out on the slide in such a
concentration that they are adequately separated from one another
(ii)
the bacterial are not washed off from the slide during staining
(iii)
bacterial form is not distorted
A chemical to be used as a stain for biological material must have at least two
properties;
(i)
it should be intensely chromogenic (colored) and
(ii)
it must combine with some cellular component
When biological stains are used correctly, they enable scientists to visually
examine cells with a considerable level of resolution and definition. Simple stains
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are used as general, all-purpose stains. They are usually basic dyes (a salt with
the color in the positive ion) that will stain the cell membranes of most bacteria
(cell membranes are negatively charged). It is usually necessary to expose the
cells to the stain for only a short period of time, during which the positively charged
stain will bind with the negatively charged cell membrane. The excess stain is
then removed by rinsing before examining the cells with a microscope. Simple
stains stain biological materials indiscriminately.
By modifying the staining procedure, using special stains, or adding chemicals, it
is possible to differentially stain bacteria. In some cases, only certain types of cells
will be stained; in other cases, only certain parts of cells will be stained. The
procedures to stain different cells or components different colours, are referred to
a differential staining procedure. Once bacteria have been properly stained, it is
usually an easy matter to discern their overall shape. Bacterial morphology is
usually uncomplicated and limited to one of a few variations.
Structural and morphology
Bacteria and archaea are prokaryotes - unicellular microbes that lack nuclei.
Bacterial cell walls are composed of a polysaccharide called peptidoglycan,
though some bacteria lack cell walls. The cell walls of archae a lack peptidoglycan
and instead are composed of other polymers. Members of both groups reproduce
asexually. Most archaea and bacteria are much smaller than eukaryotic cells.
There are various sizes, shape and arrangement of bacteria cells. Most of bacteria
are within a range of 0.20 – 2.0 µm in diameter and from 2.0 – 8.0 µm in length.
They are either coccus (spherical shaped), bacillus (rod shape) or spiral in shape
(Figure 1). Cocci are usually round but can be oval, elongated or flattened.
Diplococci consist of two cocci; streptococci consist of a chain of 3 – 6 cells of
cocci; staphylococci consist of a bunch or a cluster of cocci; tetrads consist of four
cells of cocci and sarcinae consist of eight cells of cocci. The arrangement of
bacilli can exist as diplobacilli (two bacilli), streptobacilli (a chain of 3 – 6 cells of
bacilli) or as a single bacillus cell. Spiral bacterial cells have one or more twist and
they never in straight form. Curved bacteria are called vibrios. Those with helical
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shape and fairly rigid bodies are called spirilla, while those which are helical and
flexible are called spirochaetes.
The most common characteristic of the bacterial arrangement are shown in Figure
2. Besides the three basic shapes, there are star shaped cells (Genus Stella),
square shaped (halophilic archaeobacteria, Arcula) and triangular cells (Genus
Haloarcula). The shape of bacteria is determined by heredity and most bacteria
are monomorphic (maintain a single shape). Nevertheless, a number of
environmental conditions can alter that shape. As for genus such as Rhizobium,
Bradyrhizobium and Corynebacterium which are genetically pleomorphic can have
many shapes, not just one shape.
Figure 1: The most common bacterial shapes
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Figure 2: The most common characteristic of bacterial arrangement
Fungi are organisms whose cells are eukaryotic, that is, each of their cells
contains a nucleus composed of genetic material surrounded by a distinct
membrane. Fungi are different from plants because they obtain their food from
other organisms (rather than making it by photosynthesis). They differ from
animals by having cell walls. Fungi can be found in two forms that are yeast
(unicellular and budding cells) and multicellular filamentous or also known as
molds.
Yeast is a unicellular with a single nucleus and can be found in the form of
spherical or ovoid cells that reproduce asexually by budding or binary fission.
However yeast cells can reproduce sexually by spore formation. On the other
hand, the filamentous fungi or molds are velvety like of fungi that produce longbranched hyphae either coenocytic or septate. A mass of hyphae is called
mycelium. Filamentous fungi or molds reproduce asexually by means of spores or
conidia, chlamydospores, arthrospores, blastospores and sporangiospores. The
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spores and conidia are generally formed on the tips of hyphae and these can be
easily dislodge and distributed. The sexual reproduction of fungi involves the union
of two compatible nuclei which finally form sexual spores such as oospores,
zygospores, ascospores or basidiospore.
Figure 3: Type of colony growth
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In 1884 the Danish bacteriologist Christian Gram developed a staining technique
that separates bacteria into two groups: those that are gram-positive and those
that are gram-negative. The procedure is based on the ability of microorganisms
to retain the purple color of crystal violet during decolorization with alcohol. Gramnegative bacteria are decolorized by the alcohol, losing the purple color of crystal
violet. Gram-positive bacteria are not decolorized and remain purple. After
decolorization, safranin, a red counterstain, is used to impart a pink color to the
decolorized gram-negative organisms.
4.0 MATERIALS AND EQUIPMENTS
4.1
Cultures of bacteria, yeast and mould will be provided as follows:
Bacillus spp. on NA or PCA
Escherichia coli on VRBA
Saccharomyces cerevisiae on MEA
Aspergillus niger
4.2
Gram staining chemicals
4.3
Methylene Blue Stain.
4.4
Bunsen burner/Spirit lamp.
4.5
Inoculation loop.
4.6
Compound microscope, slides and coverslips.
6.0 PROCEDURES
EXPERIMENT 2A:
5.1 WET MOUNT PREPARATION
5.1.1
Place a drop of water on a clean piece of slide
5.1.2
Transfer some of the cultures with an inoculation loop. Mix it into
the drop of water on the slide until a suspension is achieved. A cell
suspension may be initially prepared and methylene blue stain may
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be added.
5.1.3
Then, place the cover slip onto the suspension without introducing
bubbles.
5.1.4
Place the slide with specimen onto the stage of the microscope.
Observe the specimen using various magnifications as advised.
Record the size and shape of the microorganism where
appropriate.
Figure 4: Preparation of a wet mount
5.2 SMEAR PREPARATION
5.2.1
Place a drop of sterile distilled water on a clean glass slide
5.2.2
Transfer a small sample of a colony into the drop of water and mix it
gently. Spread the emulsion evenly on the surface of a glass slide
to give a relatively thin and smooth layer
5.2.3
Allow the smear to dry by normal evaporation of the water.
5.2.4
Once the smear is completely dry, pass the slide over the flame of
a Bunsen burner to heat-kill and fix the microorganism to the slide.
Notes: If a slant or plate is used, a small amount of bacterial/fungal growth is
transferred to a drop of water on a glass slide and mixed. If the medium is
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liquid, place one or two loops of the medium directly on the slide and spread
the bacteria over a large area as illustrated in Figure 3.
Figure 5: Smear preparation
5.3 SIMPLE STAINING
5.3.1
Prepare smears of bacterial and fungal as previously described
previously.
5.3.2
Cover the smear with sufficient methylene blue and/or cotton blue
stain for 1 minute.
5.3.3
Rinse the stain off with tap water.
5.3.4
Remove the slide from the rack and allow excess water to drain off
by holding the slide vertically on a tissue.
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Examine the film under the microscope. If you examine using the
high power objective (100x), place a drop of immersion oil on the
film and cover with a cover slip.
EXPERIMENT 2B:
5.4 GRAM STAINING
5.4.1
Prepare smears of bacterial culture as described previously.
5.4.2
Flood the smear with Gram’s crystal violet for 20 seconds
5.4.3
Briefly wash off the stain by rinsing gently with water.
5.4.4
Then flood the smear with Gram’s iodine for 30 seconds
5.4.5
Pour off the iodine and wash thoroughly with acetone or alcohol.
This step is critical. Thick smears will need more time than thin
ones. Decolorization has occurred when the solvent flows
colorlessly from the slide.
5.4.6
Wash the acetone gently with running tap water
5.4.7
Cover the smear with safranin for 30 seconds
5.4.8
Wash the safranin gently for a few seconds, blot and dry the smear
at room temperature
5.4.9
The slide may be examined under a microscope. If you examine
using the high power objective (100x), place a drop of immersion oil
on the film and cover with a cover slip.
5.4.10
The results with purple or purplish black bacteria indicates Gram
positive, while a red or pink indicates Gram negative.
6.0 RESULTS AND DISCUSSIONS
Observe under the microscope the plates and slide to describe the gross
morphology in terms of colour, appearance and/or the sporangia. Draw and label
the morphology. Tabulate the observation according to the format below where
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appropriate (Table 1). Take note that some descriptions used may not be suitable/
applicable for a particular microorganism. Your trainer will advise you accordingly.
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Table 1: Appearance and morphology of microorganisms studied.
Microorganism
Appearance
(Draw a few
representative cells)
Morphology
Shape
Arrangement
Yeast
Fungus
Bacterium
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Single colony on plate
Elevation
Margin
ERT 107- Microbiology for Bioprocess Engineering
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7.0 REFERENCES
7.1
Bauman, R. 2007. Microbiology with diseases by taxonomy 2nd Edition.
Pearson Education, Prentice Hall.
7.2
Garbutt, J.W. & Bartlett, A.J. 1972. Experimental Biology with MicroOrganisms: Teachers’ Guide. Butterworth & Co, London.
7.3
Harley, J.P. 2007. Laboratory exercises in Microbiology 7th Edition.
McGraw Hill, New York.
7.4
Messley,
K.E.
& Norrell,
S.A. 2003.
Principles and Application:
Microbiology Laboratory Manual. Prentice Hall, Upper Saddle River.
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EXPERIMENT 3
TOTAL CELL COUNT USING HAEMOCYTOMETER
1.0
OBJECTIVE
1.0
To familiarize with hands-on experiment in microbial studies.
1.1
To conduct total cell count using Haemocytometer
2.0 COURSE OUTCOME
CO1: Ability to acquire and apply knowledge of microbiology concepts and
techniques in bioprocess
3.0 INTRODUCTION
The haemocytometer is a thick glass slide originally designed for counting
cells in blood samples (haemocytes). The counting areas are in the central area of
the slide and consist of accurately ruled squares of fixed dimensions, the platform
is a fixed depth. Hence, each square is a known volume of solution. Therefore, by
counting the cells in a square, you can calculate the number of cells/volume.
Haemocytometer chamber is normally used to count cell/bacteria number or
spores of moulds.
3.0
MATERIALS AND EQUIPMENTS
3.1
Heamocytometer
4.0 PROCEDURES
4.1
TOTAL CELL COUNT USING HAEMOCYTOMETER
Figure 9 and Figure 10 shows a diagrammatic sketch of a haemocytometer
chamber. Before proceeding with the counting, make sure you have thoroughly
understand the counting technique when using the chamber. A good
understanding of the procedure will help you to calculate the concentration of the
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cells or spores in the chamber and subsequently, in the suspension of cells
supplied. The procedures are as follows:
4.1.1
Ensure that the slide and the cover slip are free of oil and grease.
4.1.2 Cleaning the slide with xylol and drying will ensure they are free of oil and
grease before use.
4.1.3
Place the bacteria suspension into the chamber with the micropipette.
Using a pasteur pipette, touch a drop of cell suspension to the edge of the
cover slip (x marks the spot) (Figure 9) and the liquid will spread under
the cover slip by surface tension. You only need to fill one counting
platform.
4.1.4
Alternatively, prior to applying the suspension, the cover slip is placed on
the slide and by pressing gently a so-called Newton ring is formed between
the slide and cover slip. The bacteria suspension is flooded into the
chamber and excess fluid is removed when the cover slip is pressed
against the slide.
4.1.5
Subsequently the slide is placed onto the stage of the microscope for
examination and enumeration.
4.1.6
Observe under moderate magnification and control the intensity of the
light. Observe also under the oil immersion objective.
4.1.7
Count the number of cells in 5 squares. Dilution of the suspension may
need to be carried out if the cell number is too many. A good estimation is
when between 2 – 5 cells are present in every small squares.
4.1.8
The suspension on the slide must not be allowed to stand too long, as
the volume may change and jeopardize the accuracy of the count.
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Figure 9: Illustration of the Haemocytometer with Plan View and Side View
Figure 10: A diagrammatic representation of the counting grid of the
haemocytometer
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Figure 11: A diagrammatic representation of the internal dimensions counting grid
of the haemocytometer
Counting Chamber
The depth of the counting chamber is 0.2 mm and the small squares are 0.25 mm
x 0.25 mm. There are 9 sets of 16 small squares. Ideally and practically, you
should count 100 -200 cells in total in the suspension. In order to avoid counting
a cell twice, use the following procedure. If cell lies across a line, on a North
or West direction of a small square, count it. However, if it lies on a South or
East direction, then and ignore it.
Quick Estimate: Assuming there are sufficient cells (50 -100) in 8 squares, count
the number of cells in 8 small squares and multiply this number by 104.This gives
the number of cells /ml, i.e. 100 cells in 8 squares = to a cell concentration of 106
cells/ml.
5.0 RESULTS AND DISCUSSION
Observe under the microscope the haemocytometer to record the count of
microorganisms cells.
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EXPERIMENT 4
DETERMINATION OF BACTERIAL GROWTH CURVE
1.0 OBJECTIVES
1.1
To understand the growth dynamics of a bacterial culture.
1.2
To identify the typical phases of a bacterial growth curve
1.3
To plot a growth curve and determine the generation time of a culture
2.0 COURSE OUTCOME
CO2: Ability to conduct investigation into complex problems as well as to
analyze and interpreted data related to metabolism, growth kinetic and
fermentation of microbes
3.0 INTRODUCTION
The four phases (lag, logarithmic, stationary, and death or decline) of growth of a
bacterial population can be determined by measuring the turbidity of the
population in a broth culture. Turbidity is not a direct measure of bacterial numbers
but an indirect measure of biomass, which can be correlated with cell density
during the log growth phase. Since about 107 bacterial cells per milliliter must be
present to detect turbidity with the unaided eye, a spectrophotometer can be used
to achieve increased sensitivity and obtain quantitative data.
The construction of a complete bacterial growth curve (increase and decrease in
cell numbers versus time) requires that aliquots of a shake-flask culture be
measured for population size at intervals over an extended period. The bacterial
population will be plotted by using both an indirect and direct method for the
measured of growth. The resulting growth curve can be used to delineate stages
of the growth cycle. It also makes possible the determination of the growth rate of
a particular bacterium under standardized conditions in terms of its generation
time – the time required for a bacterial population to double.
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The indirect method uses spectrophotometric measurement of the developing
turbidity in a bacterial culture taken at regular intervals. These samples serve as
an index of increasing cellular mass. The graphical determination of generation
time is made by extrapolation from the log phase, as illustrated in Figure 1.
Figure 1: A typical microbial growth curve
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For example, select two points (0.2 and 0.4) on the absorbance (A) scale that
represents a doubling of turbidity. Using a ruler, extrapolate by drawing a line
between each absorbance on the ordinate, and the plotted log or exponential
phase of the growth curve. From these two data points, draw perpendicular lines
to the time intervals on the abscissa. From these data, the generation time can be
calculated as follows:
Generation time = t (A of 0.4) – t (A of 0.2)
Generation time = 90 – 60 minutes
= 30 minutes
The same graphical generation time determination can be done with a plot of
population counts.
The growth rate constant can also be determined from the data. When the log 10 of
the cell numbers or absorbance is plotted versus time, a straight line is obtained,
the slope of which can be used to determine the value of g and k. The dimensions
of k are reciprocal hours or per hour. The growth rate constant will be the same
during exponential growth regardless of the component measured (e.g., cell
biomass, number). The growth rate constant provides the microbiologist with a
valuable tool for comparison between different microbial species when standard
growth and environmental conditions are maintained.
Once the growth rate constant is known, the mean generation time (doubling time)
can be calculated from the following equation:
g= 1
k
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This equation also allows one to calculate the growth rate constant from the
generation time. As mentioned previously, the generation time can be read directly
from the bacterial growth curve plot, and the growth rate constant then
determined. To calculate the generation time (g) from these data with an equation,
use the following formula:
Generation time =
0.301t
log10Nt – log10N0
where N0 = bacterial population at point B or any other point at the beginning of
the log phase
Nt = bacterial population at point b or any other point at or near the end of
the log phase
t = time in minutes between b and B (Figure 1)
From the previous equation, one can also determine the specific mean growth rate
constant (k) for any culture during unrestricted growth. During this time, the rate of
increase of cells is proportional to the number of cells present at any particular
time. In mathematical terms, the growth rate is expressed as
k= n
t
where n is the number of generations per unit time. The symbol k represents the
mean growth rate constant. Converting the equation to logarithms:
k = log Nt – log N0
0.301t
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4.0 MATERIALS AND EQUIPMENTS
4.1
10- to 12- hours tryptic soy broth cultures of E. coli
4.2
Tryptic soy broth
4.3
Spectrophotometer
4.4
Incubator (orbital shaker)
5.0 PROCEDURES
5.1
Place 190 ml of tryptic soy broth in 500 ml conical flask.
5.2
Zero the spectrophotometer at 550 nm with the tryptic soy broth from the
flask.
5.3
Place the flask containing the tryptic soy broth in the incubator for 15
minutes at 37°C.
5.4
While slowly agitating the flask in the incubator, inoculate it with 10 ml of E.
coli culture.
5.5
Read and record the absorbance (A) of this initial culture (0 time) and
every 15 minutes thereafter for 2 hours. Be sure to suspend the bacteria
thoroughly each time before taking a sample.
6.0 RESULTS AND DISCUSSIONS
Based on your data on absorbance, complete the following table:
Construct a growth curve by plotting A against incubation time. Calculate the
mean generation time and growth rate constant using both equations and the
graphical method.
7.0 REFERENCES
7.1 Harley, J.P. 2007. Laboratory exercises in Microbiology 7th Edition. McGraw
Hill, New York.
7.2 Messley, K.E. & Norrell, S.A. 2003. Principles and Application: Microbiology
Laboratory Manual. Prentice Hall, Upper Saddle River.
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EXPERIMENT 5
ANTIMICROBIAL ASSAY
1.0 OBJECTIVES
1.1 To evaluate the effectiveness of several antibiotics, antiseptics and
antimicrobics.
1.2 To perform the Kirby-Bauer method for determination of antibiotic, antiseptics
and antimicrobics sensitivity.
1.3 To correctly interpret a Kirby-Bauer plate.
2.0 COURSE OUTCOME
CO3: Ability to evaluate and formulate the solutions of engineering problem
related to the application of microbes in the bioprocess industry.
3.0 INTRODUCTION
One method that is used to determine antibiotic susceptibility is the sensitivity disk
method of Kirby-Bauer. In this method, antibiotics are impregnated onto paper
disks and then placed on a seeded Mueller-Hinton agar plate using mechanical
dispenser or sterile forceps. The plate is then incubated for 16 to 18 hours, and
the diameter of the zone of inhibition around the disk is measured to the nearest
millimeter. The inhibition zone diameter that is produced will indicate the
susceptibility or resistance of a bacterium to the antibiotics. Antibiotic susceptibility
patterns are called antibiograms. Antibiograms can be determined by comparing
the zone diameter obtained with the known zone diameter size for susceptibility.
For example, a zone of certain size indicates susceptibility, zones of a smaller
diameter or no zone at all show that bacterium is resistant to the antibiotic.
Frequently one will see colonies within the zone of inhibition when the strain is
antibiotic resistant.
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Many factors are involved in sensitivity disk testing and must be carefully
controlled. These include size of the inoculum, distribution of the inoculum,
incubation period, depth of the agar, diffusion rate of the antibiotic, concentration
of antibiotic in the disk, and growth rate of the bacterium. If all of these factors are
carefully controlled, this type of testing is highly satisfactory for determining the
degree of susceptibility of a bacterium to a certain antibiotic.
The Kirby-Bauer method is not restricted to antibiotics. It may also be used to
measure the sensitivity of any microorganism to a variety of antimicrobial agents
such as sulfonamides and synthetic chemotherapeutics.
4.0 MATERIALS AND EQUIPMENTS
4.1
Cultures of bacteria as follows:
Escherichia coli
Staphylococcus epidermis
Bacillus subtilis
Pseudomonas aeruginosa
4.2
Several different types of antibiotics, antiseptics and/or antimicrobics.
4.3
Nutrient agar plate
4.4
Sterile swabs
4.5
Petri dish containing sterile filter paper disk
4.6
Forceps
4.7
Bunsen burner
4.8
70% alcohol
4.9
Marker pen
4.10
Metric rulers
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5.0 PROCEDURES
5.1
Obtain one of Nutrient agar plate. By using a marker pen, mark the lid of
Nutrient agar plate into four sections and label with your name, date and
the name of the bacterium to be inoculated. You are required to inoculate
the surface of the Nutrient agar plates with the test organism.
5.2
Immersed a sterile swab into a broth culture of the test organism. Use a
separate, sterile cotton swab for each bacterium.
5.3
The swab is then taken and streaked on the surface of the Nutrient agar
plate three times, rotating the plate 60° after each streaking. Finally, run
the swab around the edge of the agar. This procedure ensures that the
whole surface has been seeded.
5.4
Allow the culture to dry on the plate for 10 minutes.
5.5
Repeat steps 5.1 to 5.4 for each organism being used.
5.6
Using sterile forceps pick up one of the sterile filter-paper disks. Place
20 µl of antibiotic or antiseptic on the disk and allow it to absorb the
solution.
5.7
Place the disk in the designated area on the inoculated petri plate. Be sure
to gently press down the disc on the agar surface using sterile forceps.
This will ensure better adherence to the surface. Do not press the disk into
agar, and do not move the disk once it is placed on the agar.
5.8
Repeat steps 5.6 to 5.7 for each of the test solutions on each of your
plates, being sure to re-sterilize forceps before each application.
5.9
Incubate the plates for 24 hours at 35°C. Do not invert the plates.
5.10
Measure the zones of inhibition to the nearest mm for each of the
antibiotics or antiseptics tested. Record the results.
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6.0 RESULTS AND DISCUSSIONS
Based on your measurements, complete the following table on the susceptibility of
each test bacterium to the antibiotic by using an R (resistant), I (intermediate), or S
(sensitive):
7.0 REFERENCES
7.1 Harley, J.P. 2007. Laboratory exercises in Microbiology 7th Edition. McGraw
Hill, New York.
7.2 Messley, K.E. & Norrell, S.A. 2003. Principles and Application: Microbiology
Laboratory Manual. Prentice Hall, Upper Saddle River.
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Table 1: Susceptibility of test bacterium to the antibiotic, antiseptic and antimicrobic
_ 8
_
_
E. coli
Antibiotic or antimicrobic
Zone
S. epidermis
S, I, R
Zone size
B. subtilis
S, I, R
Zone size
P. aeruginosa
S, I, R
Zone size
S, I, R
size
_
1
_
2_
_
3
_
4
6
5_
_
6
_
7
_
8_
_
_
From the above table, which antibiotic (antimicrobic) would you use against each of the test bacteria?
_
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