Laboratory Manual in General Microbiology For Undergraduate Students., Short

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at 20
13 [ ‫]االحياء الدقيقة العملي‬
A lnaim
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Dr.S
Al-Hussein Bin Talal University

Biology department Biology department
[Laboratory Manual in General Microbiology]
hghp
Prepared by
Dr. Sulaiman Alnaimat
Mr. Saqer AbuShattal
September 2012
0
Laboratory Manual in General Microbiology
[Laboratory Manual in General Microbiology]

[ ‫]االحياء الدقيقة العملي‬
Prepared by
Dr. Sulaiman Alnaimat
Mr. Saqer AbuShattal
September 2012
|Page1
Table of contents
Table of contents ............................................................................................................................. 2
Some Microbiological Equipments ................................................................................................ 3
Microbiology Lab Practices and Safety Rules ................................................................................ 4
The Control of Microbial Growth ................................................................................................... 5
Microbiological Culture Media Preparation .................................................................................. 7
Isolation of Microorganisms from anEnvironment of Choice ...................................................... 10
Culture Transfer Instruments, Techniques, and Isolation of Pure Cultures .................................. 12
Microscopic Techniques ............................................................................................................... 18
Bacterial Morphology and Staining .............................................................................................. 27
Preparation of smear ..................................................................................................................... 29
Simple Staining (direct and indirect staining) ............................................................................. 31
Differential stain ( Gram Stain & Acid Fast Stain)....................................................................... 35
Structure stain ( Endospores stain) ........................................................................................... 39
Determination of Bacterial Numbers ............................................................................................ 42
Effects of Chemical Agents on Bacteria: ...................................................................................... 45
REFERENCES ............................................................................................................................. 47
|Page2
Bunsen burner Laminar air flow Compound Microscope

chamber
Water bath Autoclave Refrigerator
Incubator Balances Magnetic stirrer
|Page3
1. Wash your hands with disinfectant soap when you arrive at the lab and again before you
leave.
2. Absolutely no food, drinks, chewing gum, or smoking is allowed in the laboratory. Do not
put anything in your mouth such as pencils, pens, labels, or fingers. Do not store food in
areas where microorganisms are stored.
3. Purchase a lab coat and safety glasses, bring them to class, and use them.
4. Avoid loose fitting items of clothing. Wear appropriate shoes (sandals are not allowed) in the
Laboratory
5. Keep your workspace free of all unnecessary materials.
6. Disinfect work areas before and after use with 70% ethanol or fresh 10% bleach. Laboratory
7. equipment and work surfaces should be decontaminated with an appropriate disinfectant on a
8. routine basis, and especially after spills, splashes, or other contamination.
9. Label everything clearly
10. Replace caps on reagents, solution bottles, and bacterial cultures. Do not open Petri dishes in
the lab unless absolutely necessary.
11. Inoculating loops and needles should be flame sterilized in a Bunsen burner before you lay
them down.
12. Turn off Bunsen burners when is not in use. Long hair must be restrained if Bunsen burners
are in use.
13. Sterilize equipment and materials.
14. Consider everything a biohazard. Do not pour anything down the sink. Autoclave liquids and
broth cultures to sterilize them before discarding.
15. Dispose of broken glass in the broken glass container.
16. Dispose of razor blades, syringe needles, and sharp metal objects in the “sharps” container.
17. Report all injuries or accidents immediately to the instructor, no matter how small they seem
‫ قبل الدخول المختبر ويجب غلق المعطف‬Laboratory coat ‫ارتداء المعطف النظيف‬- 1
‫الحضور الى المختبر في الموعد المحدد‬- 2
‫عدم األكل والشرب أو جلب األغراض الشخصية داخل المعمل‬- 3
‫االنتباه لشرح التجارب المعملية وتنفيذها بدقة‬- 4
‫(قبل وبعد العمل‬70% ethanol (‫ بالمطهر المناسب‬Bench ‫تنظيف طاولة العمل‬- 5
‫يجب إبالغ المشرف في حال تلوث او انسكاب أي مادة او كسر أي اداة زجاجية‬- 6
‫كتابة جميع البيانات التوضيحية على كل عينة‬- 7
‫ الحرص على نظافة وسالمة االجهزة والمعدات‬-8
‫ غسل اليدين جيدا بالماء والصابون قبل مغادرة المعمل‬-9
‫ عدم لمس العينين او استخدام الفم اثناء العمل داخل المختبر‬-11
‫ كافة ادوات المختبر المستخدمة من أنابيب وماصات وشرائح توضع في االواني الخاصة بها لحين تنظيفها‬-11
‫ الشعر الطويل يجب أن يربط للخلف لتالفي خطر االحتراق والتلوث‬-12
‫ او االبرة الناقلة قبل وبعد االستعمال‬Loop ‫ تحرق ابرة التلقيح‬-13
‫ ويجررب‬,‫ يعتبررر الصررديق المصرراحب لطالررب علررم االحيرراء الدقيقررة فيجررب صرريانتة والتعامررل معررة بدقررة‬Microscope ‫ المجهررر‬-14
‫تنظيف العدسات وإزالة اثار زيت السيدر وعدم ترك الشريحة على المجهر وغلق المجهر بعد االنتهاء من الفح‬
‫ عدم رمي المواد التالفة واألوساخ في حوض الغسيل‬-15
‫ |الحرص على اطفاء اللهب بعد االنتهاء من العمل‬P
-18
age4
Introduction
The control of microbial growth is necessary in many practical situations, and significant
advances in agriculture, medicine, and food science have been made through study of this area of
microbiology.
"Control of growth", as used here, means to prevent growth of microorganisms. This control is
effected in two basic ways:
 By killing microorganisms or
 By inhibiting the growth of microorganisms.
Control of growth usually involves the use of physical or chemical agents which either kill or
prevent the growth of microorganisms. Agents which kill cells are called cidal agents; agents
which inhibit the growth of cells (without killing them) are referred to as static agents. Thus the
term bactericidal refers to killing bacteria and bacteriostatic refers to inhibiting the growth of
bacterial cells. A bactericide kills bacteria, a fungicide kills fungi, and so on.
Sterilization is the complete destruction or elimination of all viable organisms (in or on an

object being sterilized). There are no degrees of sterilization: an object is either sterile or not.
Sterilization procedures involve the use of heat, radiation or chemicals, or physical removal of
cells.
Methods of Sterilization
Heat: most important and widely used..

1. Incineration: burns organisms and physically destroys them. Used for needles ,
inoculating wires, glassware, etc. and objects not destroyed in the incineration process.
2. Boiling: 100°C for 30 minutes. Kills everything except some endospores. To kill
endospores, and therefore sterilize the solution, very long or intermittent boiling is
required.
3. Dry heat (hot air oven): 160°C/2hours or 170°C/1hour. Used for glassware, metal, and
objects that won't melt.
4. Pasteurization : s a process of heating a food, which is usually a liquid, to a specific

temperature for a predefined length of time and then immediately cooling it after it is
removed from the heat. This process slows spoilage due to microbial growth in the food.
Unlike sterilization, pasteurization is not intended to kill all micro-organisms in the food.
Instead, it aims to reduce the number of viable pathogens so they are unlikely to cause
disease
|Page5
5. Autoclaving (steam under pressure or pressure cooker): 121°C for 15 minutes (15
Ib/sq pressure). Good for sterilizing almost anything, but heat-labile substances will be
denatured or destroyed.
Antiseptics: Microbicidal agents

harmless enough to be applied to
the skin and mucous membrane.
Disinfectants: Agents that kill

microorganisms, but not
necessarily their spores, not safe
for application to living tissues;
they are used on inanimate
objects such as tables and floors
Figure 1 : An autoclave is a device used to
sterilize equipment and supplies
Irradiation: usually destroys or distorts nucleic acids. Ultraviolet light is usually used
(commonly used to sterilize the surfaces of objects), although x-rays and microwaves are
possibly useful.
Filtration: involves the physical removal (exclusion) of all cells in a liquid or gas, especially
important to sterilize solutions which would be denatured by heat (e.g. antibiotics, injectable
drugs, amino acids, vitamins, etc.)
Chemical and gas:

Alcohol 70% : Antiseptics and disinfectants
Formalin:( Formaldehyde)is an organic compound with the formula CH2O, used as disinfectants
Ethylene oxide gas : This highly reactive gas (C2H4O) is flammable, toxic, and a strong mucosal
irritant, used for contaminated medical tools.
Halogens: Chlorine, iodine, and derivatives of these halogens are suitable for use as
disinfectants. Chlorine and iodine show a generalized microbicidal effect and also kill spores
Irradiation (microwave, UV, x-ray, Gamma radiation): destroys microorganisms as described

under sterilization. Many spoilage organisms are easily killed by irradiation.
Preservatives: static agents used to inhibit the growth of microorganisms, most often in
foods. If eaten they should be nontoxic
|Page6
Culture media (singular, medium) are the nutrient solutions used to grow microorganisms in
the laboratory.
Nutrition materials in media :

1. Water : used to dissolved materials to be transported across the cytoplasmic membrane
2. Source of carbon: required for the construction of all organic molecules, usually glucose
3. Source of nitrogen : obtained from an inorganic source or from an organic source e.g.
proteins , broken down into amino acids, or beef extract broken to nucleic acid.
4. Buffer system: most bacteria can growth at 7.0 pH. This is achieved by using a buffer
system e.g. carbonate or phosphate buffer.
5. Source of minerals : required in small amounts, e.g. Iron, Sulfur and phosphorous.
The physical forms of the culture media are used: liquid, or broth, media; semisolid
media; and solid media. The major difference among these media is that solid (1.5-1.8% agar)
and semisolid ( ˂ 1.0% agar ) media contain a solidifying agent (usually agar), whereas a liquid
medium does not.
Liquid media, such as nutrient broth, tryptic soy broth, or brain-heart infusion broth , can
be used to propagate large numbers of microorganisms in fermentation studies and for various
biochemical tests.
Semisolid media can also be used in fermentation studies, in determining bacterial motility,
and in promoting anaerobic growth. Solid media, such as nutrient agar or blood agar, are used (1)
for the surface growth of microorganisms in order to observe colony appearance, (2) for pure
culture isolations, (3) for storage of cultures, and (4) to observe specific biochemical reactions.
Solid media can be poured into either a test tube or Petri plate (dish). If the medium in the test
tube is allowed to harden in a slanted position, the tube is designated an agar slant ; if the tube is
allowed to harden in an upright position, the tube is designated an agar deep tube and if the agar
is poured into a Petri plate, the plate is designated an agar plate
Agar : is a gelatinous polymer substance derived

from red algae. Solid at room temperature,
remaining firm at temperature as high as 65°C.
Agar melts at approximately 85°C, a different
temperature from that at which it solidifies, 32-
40°C.
Figure2 : The different types of Solid media

|Page7
Classes of Culture Media ( Composition)
1. Defined media are prepared by adding precise amounts of highly purified inorganic or
organic chemicals to distilled water. Therefore, the exact composition of a defined
medium is known.
2. Complex media (undefined media) employ digests of microbial, animal or plant

products, such as casein (milk protein), beef (beef extract), soybeans ( tryptic soy broth),
yeast cells (yeast extract), or any of a number of other highly nutritious yet impure
substances. its exact composition is unknown.
3. An enriched medium, often used for the culture of otherwise difficult-to-grow

nutritionally demanding(fastidious) microorganisms, starts with a complex base and is
embellished with additional nutrients such as serum, blood, or other highly nutritious
substances.
Classes of Culture Media ( Purpose)

1. A selective medium contains compounds that inhibit the growth of some
microorganisms (antibiotics) but not others. For example, media are available for the
selective isolation of pathogenic strains of E. coli from food products
2. differential medium is one in which an indicator, typically a reactive dye, is added that
reveals whether a particular chemical reaction has occurred during growth. Differential
media are quite useful for distinguishing different species of bacteria and are therefore
widely used in clinical diagnostics and systematic microbiology, e.g. blood agar
*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*+*
Procedure: Media preparation
We will make some broth and some agar media according to the directions below:
 Preparing a broth Medium

1. The dehydrated media is weighted and dissolved in a specific amount of water,
pH is checked and adjusted according to the manufacturer's instructions. Measure
the materials to make 100 ml of broth and place them in a 250 ml flask.
2. Boil the media using heat until the medium appears clear, the dispense in test
tubes ( 5 or 10 ml).
3. Autoclave at 121°C under 15 Ibs pressure for 15 min.
 Preparing a Agar Medium

1. The dehydrated media is weighted and dissolved in a specific amount of water, pH is
checked and adjusted according to the manufacturer's instructions. Measure the
materials to make 500 ml of agar and place them in a 1000 ml Duran bottle.
2. Bring to a gentle boil on a hotplate until all ingredient are dissolved.
3. Autoclave at 121°C under 15 Ibs pressure for 15 min.
|Page8
 Procedure for Autoclaving

1. Your instructor will demonstrate the use of the autoclave.
2. Load the autoclave with the freshly prepared culture media.
3. Close and lock the autoclave door.
4. Set the autoclave time for 15 minutes or longer and select a slow rate of exhaust.
5. Make certain that the autoclave temperature is set to 121°C.
6. Start the autoclave by pushing the start button or twisting the knob to the start
position. When the period of sterilization is completed and the pressure in the
chamber reads, carefully open the door and remove the containers, using heat-proof
gloves.
Aseptic technique : refers to a procedure that is performed under sterile conditions.
 Procedure for Pouring autoclaved culture media

1. After autoclaving period, cool the flask or Duran bottle to 50 °C.
2. Open the cover of the Petri dish halfway and keep the lid pointed down.
3. Working quickly, remove the cap of the flask, flame the mouth, and
pour the liquid agar into a sterile Petri dishes while the cover is partially raised.
4. Allow all plates to cool for about 15 minutes without moving.
Figure 3 : pouring a plate
|Page9
Objective
In order to demonstrate the ubiquity and diversity of microbes in the environment, samples from
immediate areas of the environment and/or from your body will be obtained and cultured.
Introduction
Microorganisms are found throughout the environment: in the air and water; on the surface of
any object such as clothes, walls, furniture; in soil and dust; and on and in our own bodies (skin
and mucous membranes). Microbes are even found in extreme, seemingly inhospitable
environments: living and thriving microbes are common in hot springs with temperatures in
excess of 95°C, in Antarctic seawater below 0°C, and in acid mine waters whose pH is below 4.
Ordinarily these ubiquitous microorganisms are of no threat to healthy humans, and quite a few
are actually beneficial. Normal flora on and in our body act as an initial defense against invading
pathogens. Normal flora compete with foreign microorganisms for nutrients and space, thereby
preventing them from flourishing. In the absence of the normal flora (e.g., due to prolonged
antibiotic treatment), opportunistic microorganisms may become established, increasing the risk
of developing disease. In the environment, many organisms in soil play important geochemical
roles in processing vital elements such as phosphorus and nitrogen, making them available to
other living things. Nonetheless, there are organisms in our environment that are harmful: soil
also harbors the spores of the tetanus and botulism bacteria.
Materials
Sterile culture swabs , 9 ml sterile dilution water in a test tube, nutrient agar plates, nutrient broth
and a few sterile tongue depressors for throat swabs.
Possible samples:
1. Human body (Throat, skin, scalp, teeth, ear, under the nails between toes, armpit )
2. Environment ( Garden soil, bench top (before and after disinfection), sink basin, water tap,
2222222222222222coin, doorknob, windowsill, air in the lab)
Incubator: is a device used to grow and maintain of course microbiological cultures or cell cultures. The
incubator maintains optimal temperature, humidity and other conditions such as the carbon dioxide (CO2)
and oxygen content of the atmosphere inside.
| P a g e 10
Procedure
For isolation bacteria from the garden soil
1. Place 1 g of garden soil in a 99-ml sterile water blank

2. Mix the soil and water thoroughly by shaking the water-soil mixture vigorously for 3
minutes, keeping your elbow on the lab table.
3. Transfer 1 ml of this mixture to the 2nd water blank (9 ml sterile dilution water) and mix.
4. Transfer 1 ml of this mixture to the 3rd water blank (9 ml sterile dilution water) and mix
5. Take 0.1 ml of the above suspension then spread it on the agar plate
6. Invert the Petri plates and incubate for 3 to 7 days at room temperature. Observe daily for
the appearance of colonies
Figure4 : Enumerating soil microorganisms
For isolation bacteria from other sources
1. With a marker, label the bottom of the plates and the side of the tubes with the date, the
name of the sample to be inoculated (e.g. bench top, skin), and your initials.
2. Open the “stick” end of the sterile culture swabs to avoid contamination of the swab.
Swab samples and inoculate plates with your samples. Use a fresh swab for each plate.
3. For plates, gently, so as not to mess up the agar surface, rub the swab over the entire
surface of the Petri dish without going back over areas you have already swabbed.
4. Invert the Petri plates and incubate for 3 to 7 days at room temperature. Observe daily for
the appearance of colonies.
| P a g e 11
Aseptic Technique
Because microorganisms are everywhere, culture media must be sterilized before use.
Sterilization is typically achieved with moist heat in a large pressurized chamber called an
autoclave. Once a sterile culture medium has been prepared, it is ready to receive an inoculum to
start the growth process. This manipulation requires aseptic technique, a series of steps to
prevent contamination during manipulations of cultures and sterile culture media . A mastery of
aseptic technique is required for success in the microbiology laboratory, and it is one of the first
methods learned by the novice microbiologist. Airborne contaminants are the most common
problem because the dust in laboratory air contains microorganisms. When containers are
opened, they must be handled in such a way that contaminant laden air does not enter.
Suggested procedure to use to achieve aseptic technique

1. Growth medium and its containers must be sterilized as soon as the medium is prepared.
2. The container must be covered to prevent entrance of microorganisms on dust particle
sand aerosols .
3. Always sterilize your bench before and after use.( 70% ethanol).
4. Always sterilize your inoculating loop or needle after you complete the transfer.
5. When your cultures are open do not talk.
6. Never leave the tube open any longer than the amount of time needed to transfer the
culture.
7. Never enter a culture with an inoculating loop that has not been sterilized.
8. Always flam the lips of the tube both before you insert the loop and after transfer the
culture
9. Work quickly an all transfers.
10. Never place the cap or plug on the work surface or let it touch anything except the flamed
lip and the culture tube.
11. Wash your hands before and after the laboratory session.
| P a g e 12
Table 1: Summary of the potential points of contamination and the techniques employed to minimize the risk .
Contamination Risk Precaution

Flame and cool.
Inoculating loop Do not lay down loop until procedure is complete.
Work close to Bunsen flame.
Open lid for as short a time as possible.
Opening of Petri dish (solid medium) Open lid just enough to insert wire loop.
Opening of liquid cultures (bottles, tubes) Hold the lid in crook of little finger – never place
on work bench.
Pass neck through a hot Bunsen flame before
insertion and after withdrawal of loop to kill any
contaminating organisms.
Subculturing is the aseptic transfer of micro-organisms from a culture to fresh medium by one
of the transfer instruments ( loop, needle, swab, and pipette)
Figure 5: Microbiological Transfer Instruments. (a) Inoculating needle, and (b) inoculating loop,(c) and (d)
pipette, (e) plastic pump, (F) rubber bulb.
| P a g e 13
Microbiological Transfer Instruments
1. Inoculating loops and needles:

a. Are made of Ni-Chrome or Platinum
b. Flaming is the incineration of all life forms on the loop.
c. Flaming begins at the handle and slowly continuous towards the tip. (Fig 6).
 This prevents the bacteria from forming aerosols
 Aerosols are water droplets of live bacteria sprayed into the air if you
flame beginning at the tip first.
2. Pipettes
a) Are sterilized and stored in cans.
b) Other plastic pipettes are prepackaged and are disposable.
Figure 6 : Good flaming technique. Flaming begins at the handle and

slowly continuous towards the tip, placing a loop in blue cone of flame
| P a g e 14
Techniques for isolation of pure culture:
The following are a viable techniques that used to isolate pure colonies:
1. The streak –plate method:

 In this technique, the bacterial mixture is transferred to the edge of an agar plate
with an inoculating loop and then streaked out over the surface in one of several
patterns. At some point on the streaks, individual cells will be removed from the
loop as it glides along the agar surface and will give rise to separate colonies
Again, one assumes that one colony comes from one cell. The key principle of
this method is that by streaking, a dilution gradient is established on the surface of
the plate as cells are deposited on the agar surface.
 Procedure
1. Flame the inoculating loop to red-hot, allow it to cool near burner in air.
2. Hold the your isolated culture ( previous exercise) in left hand near the flame.
Open the lid of the plate just enough to insert wire loop. Aseptically withdraw a
loopful culture with loop.
3. Place the inoculum on the agar plate at least 1 cm away from sides. Spread it in
two to three square cm areas.
4. With sterile cool needle streak or spread the culture from one corner to another
and rotating the plate by 90° after streaking 4-5 times in one direction without
overlapping previous streak as demonstrated by the instructor or shown in picture
as below:
Pure culture: laboratory

Figure 7 : The streak –plate method culture containing a single
species of organism
| P a g e 15
2. Pour plate method
 In this method we will be able to understand the pour plate method in which the
bacteria are mixed evenly distributed and separated evenly in the throughout the
liquid. The method agar is then poured into an empty plate and allowed to solidify.
After inoculation discrete bacterial colonies can then be formed growing both on the
agar media and in agar then on the isolated colonies can be a separately picked of the
isolation plate and transfer to new sterile medium. One advantage of this method is
that it requires somewhat less skill than that required for a good streak plate; a
disadvantage, however, is that it requires more media, tubes, and plates.
Figure 8 : The Pour-Plate Technique. The original sample is diluted several times to
decrease or dilute the population sufficiently. 1 ml of each dilution is then dispensed into
the bottom of a Petri plate. Agar pours are then added to each plate. Isolated cells grow
into colonies and can be used to establish pure cultures
| P a g e 16
3. Spread plate method

 In the streak plate method of isolating colonies, you diluted the bacterial culture on
the agar plate. In the spread plate method, you would dilute the bacterial culture in
tubes and then transfer the diluted samples to multiple agar plates. In a sample that is
adequately diluted, the cells will be spread far enough apart on the agar that they will
grow into individual colonies.
Figure 9: A comparison between the pour plate method and the spread plate method
 Note : students will use the spread plate method later on in this manual
| P a g e 17
Introduction
Microscopes: are instruments used to magnify objects that cannot be seen by the unaided eye(
which is unable to distinguish objects with a diameter less than 0.1 mm)
Essential features of various microscopes

1. Bright-field microscope
This is the conventional microscope which is most widely used in the field. It magnifies
images using two lens systems. Initial magnification occurs in the objective lens. Most
microscopes have at least three objective lenses on a rotating base, and each lens may be
rotated into alignment with the eyepiece or ocular lens in which the final magnification
occurs. The objective lenses are identified as the low-power, high-dry, and oil immersion
objectives. It is of particular value for examination of stained smears, especially when
used at 1000 x total magnification or greater (usually employing an oil immersion lens),
but it provides little information about internal cell structure.
2. Dark-Field microscope:
The compound microscope may be fitted with a darkfield condenser that has a
numerical aperture (resolving power) greater than the objective. Light passing through
the specimen is diffracted and enters the objective lens, whereas undiffracted light does
not, resulting in a bright image against a dark background. Since light objects against a
dark background are seen more clearly by the eye than the reverse, dark-field microscopy
| P a g e 18
t 2013
ln aima
i m an A is useful in observing unstained living microorganisms, microorganisms that are difficult
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Dr. to stain, and spirochetes, which are poorly defined by bright-field microscopy.
3. Phase-Contrast Light Microscope

Certain transparent, colorless living microorganisms and their internal organelles are
often impossible to see by ordinary bright-field or dark-field microscopy because they do
not absorb, reflect, refract, or diffract sufficient light to contrast with the surrounding
environment or the rest of the microorganism. Microorganisms and their organelles are
only visible when they absorb, reflect, refract, or diffract more light than their
environment. The phase-contrast microscope permits the observation of otherwise
invisible living, unstained microorganisms. In the phase-contrast microscope, the
condenser has an annular diaphragm, which produces a hollow cone of light; the
objective has a glass disk (the phase plate) with a thin film of transparent material
deposited on it, which accentuates phase changes produced in the specimen. This phase
change is observed in the specimen as a difference in light intensity. Phase plates may
either retard (positive phase plate) the diffracted light relative to the undiffracted light,
producing dark-phase-contrast microscopy, or advance (negative phase plate) the
undiffracted light relative to the directed light, producing bright-phase contrast
microscopy.
| P a g e 19
4. Fluorescence Microscopy
Fluorescence is the property of emitting rays having wavelength different from that of
incident rays. Object becomes luminous against dark background. Fluorescent materials
are generally of two kinds: one present naturally in cells and the other include the
induced one by staining the object with fluorescent dyes or flurochromes. In fluorescent
microscope, an object emits light when examined under UV rays. Radiations exciting the
luminosity do not contribute to image formation. Such objects absorb radiant energy and
release trapped energy when excited as visible light quickly to return to more stable state.
Two kinds of filters are used for filtering harmful rays. Excitation filters, which transmit
only the rays of visible range while blocking UV radiations the illuminating beam only
excluding the radiations, and Barrier filters prevent passing of excited radiations in
microscope to protect eyes from UV rays. The technique has become very important in
medical microbiology, microbial ecology and study of bacterial pathogenesis. The objects
are identified after staining with fluorescent dyes or flurochromes or specifically labeled
fluorescent antibodies using immunofluorescence procedures.
5. Electron Microscopy
Electron microscopes use electrons instead of visible light (photons)to image cells and
cell structures. Electromagnets function as lenses in the electron microscope, and the
whole system operates in a vacuum . Electron microscopes are fitted with cameras to
allow a photograph, called an electron micrograph, to be taken.
a. Transmission Electron Microscopy (TEM)

The transmission electron microscope (TEM) is used to examine cells and cell
structure at very high magnification and resolution. The resolving power of a TEM is
much greater than that of the light microscope, even enabling one to view structures
| P a g e 20
at the molecular level. This is because the wavelength of electrons is much shorter
than the wavelength of visible light, and wavelength affects resolution . For example,
whereas the resolving power of a high-quality light microscope is about 0.2
micrometer, the resolving power of a high-quality TEM is about 0.2 nanometer (nm,
1029 m). With such powerful resolution, even individual protein and nucleic acid
molecules can be visualized in the transmission electron microscope.
Unlike visible light, however, electron beams do not penetrate very well; even a
single cell is too thick to reveal its internal contents directly by TEM. Consequently,
special techniques of thin sectioning are needed to prepare specimens before
observing them. A single bacterial cell, for instance, is cut into many, very thin (20–
60 nm) slices, which are then examined individually by TEM. To obtain sufficient
contrast, the preparations are treated with stains such as osmic acid, or permanganate,
uranium, lanthanum, or lead salts. Because these substances are composed of atoms
of high atomic weight, they scatter electrons well and thus improve contrast.
b. Scanning Electron Microscopy

If only the external features of an organism are to be observed, thin sections are
unnecessary. Intact cells or cell components can be observed directly by TEM
with a technique called negative staining. Alternatively, one can image the
specimen using a scanning electron microscope (SEM).In scanning electron
microscopy, the specimen is coated with a thin film of a heavy metal, such as
gold. An electron beam then scans back and forth across the specimen. Electrons
scattered from the metal coating are collected and activate a viewing screen to
produce an image . In the SEM, even fairly large specimens can be observed, and
the depth of field (the portion of the image that remains in sharp focus) is
extremely good. A wide range of magnifications can be obtained with the SEM,
from as low as up to about , but only the surface of an object is typically
visualized.
| P a g e 21
Figure11 : Electron micrographs. (a) Micrograph of a thin section of a dividing bacterial

cell, taken by transmission electron microscopy (TEM). Note the DNA forming the nucleoid.
The cell is about 0.8 µm wide. (b) TEM of negatively stained molecules of hemoglobin. Each
hexagonal-shaped molecule is about 25 nanometers (nm) in diameter and consists of two
doughnut-shaped rings, a total of 15 nm wide. (c) Scanning electron micrograph of bacterial
cells. A single cell is about 0.75 µm wide
Components of the compound light microscope

1. Framework All microscopes have a basic frame structure, which includes the arm and
base. To this framework all other parts are attached.
2. Stage The horizontal platform that supports the microscope slide is called the stage.
Note that it has a clamping device, the mechanical stage, which is used for holding and
moving the slide around on the stage.
| P a g e 22
3. Light Source In the base of most microscopes is positioned some kind of light source.
Ideally, the lamp should have a voltage control to vary the intensity of light.
4. Lens Systems All microscopes have three lens systems: the oculars, the objectives, and
the condenser.
A. The ocular, or eyepiece, is a complex piece, located at the top of the instrument,
that consists of two or more internal lenses and usually has a magnification of
10×.
B. Three or more objectives are usually present. Note that they are attached to a
rotatable nosepiece, which makes it possible to move them into position over a
slide. Objectives on most laboratory microscopes have magnifications of 10× ,
45×, and 100×, designated as low power, high-dry, and oil immersion,
respectively. Some microscopes will have a fourth objective for rapid scanning of
microscopic fields that is only 4×.
C. The third lens system is the condenser, which is located under the stage. It
collects and directs the light from the lamp to the slide being studied. The
condenser can be moved up and down by a knob under the stage.
5. Focusing Knobs The concentrically arranged coarse adjustment and fine adjustment
knobs on the side of the microscope are used for bringing objects into focus when
studying an object on a slide.
| P a g e 23
Theoretical principles of microscope
 Magnification of microscope is the multiplication product of the powers of objective

and ocular lenses. The ocular lens are 10x magnification. Objective lens are 4x, 10x,
40x, 100x magnification. So once an objective lens is selected, the total magnification
would be given by its product with the 10x magnification of the ocular lens. For
example, if objective lens selected is 40x, total magnification would be:
(10x)(40x)=400x total.
 The resolution of a microscope is the ability to clearly determine two separate points,
or objects, as singular, distinguished entities. If the object are closer together than
appropriate for your resolution, they blur together, making it impossible to
differentiate.
 Refractive index (or index of refraction) n of a substance (optical medium) is a

number that describes how light, or any other radiation, propagates through that
medium. When light passes from a material of one refractive index to material of
another, as from glass to air or from air to glass, it bends. Light of different
wavelengths bends at different angles, so that as objects are magnified the images
become less and less distinct. Oil immersion objective lenses are used to further
magnify a specimen. The lens requires more light rays to pass through it since it has
more mirrors inside, requiring the use of the oil. The oil functions to refract the light
rays towards the center of the lens (the normal line of the specimen). By refracting the
light towards the center, more rays of light enter the objective, allowing you to see the
specimen in a much higher resolution (higher magnification).
| P a g e 24
Procedure for Basic Microscopy: Proper Use of the Microscope

1. Always carry the microscope with two hands. Place it on the desk with the open part
away from you.
2. Clean all of the microscope’s lenses only with lens paper and lens cleaner if necessary.
Do not use paper towels or Kimwipes; they can scratch the lenses. Do not remove the
oculars or any other parts from the body of the microscope.
3. Always focus the object by moving the Objective away from the glass slide. Avoid
focusing downwards.
4. With coarse adjustment knob move the Objective to be used until it nearly touches the
surface upper surface of the mounted specimen. Then focus by moving the coarse knob
upwards until the object comes into view. Complete the focusing with Fine adjustment
knob.
5. Adjust the mirror and light while using low power Objective to give adequate
illumination.
6. While observing unstained objects, the Iris diaphragm should be barely open to achieve
good contrast. Iris diaphragm is fully open with higher magnification and while viewing
stained objects.
7. Always, clean the lenses before and after use with lens paper. Do not touch the lenses
with hands. Leave Objectives with lowest power in working position.
8. Keep the microscope covered when not in use.
9. Observe the slides with both eyes open. Do not incline the microscope. Adjust the stool
to use the instrument comfortably.
10. Avoid direct sunlight. North light is advantageous.
11. Place a drop of immersion oil on the illuminated area of slide and shift to 100X
objective. Lower the Objective slowly viewing from sides until the lens contacts the oil
drop and then the surface of slide.
12. Prior you place the microscope in wooden box at the conclusion of each laboratory period
turn the nosepiece until the low power objective is in place and lower to the maximum
until it reaches stop.
| P a g e 25
Note: Lab work in this session is to ask students to use microscope to visualize prepared slides
or if there is enough time students might prepare a wet mounts slide as described below
Wet Mount
Used for aquatic samples (dirty water), living organisms and natural observations, wet mounts
suspend specimens in fluids such as water, brine, glycerin and immersion oil. A wet mount
requires a liquid, tweezers, pipette and paper towels.
To prepare the slide:
 Place a drop of fluid in the center of the slide
 Position sample on liquid, using tweezers
 At an angle, place one side of the cover slip against the slide making contact with outer
edge of the liquid drop
 Lower the cover slowly, avoiding air bubbles
 Remove excess water with the paper towel
Figure11 : Preparation of a wet mount slide
| P a g e 26
Introduction
Visualization of microorganisms in the living state is most difficult, not only because they are
minute but also because they are transparent and practically colorless when suspended in an
aqueous medium. A bacterium consists of a clear protoplasmic matter, which differs slightly in
refractive index from the medium in which they are growing. To study their properties and to
differentiate microorganisms into specific groups for diagnostic purposes biological stains and
staining procedures in conjunction with light microscopy have become major tools in
microbiology.
What are stains and why do they stain cells

1. Stains differentiate microorganisms from their surrounding environment
2. They allow detailed observation of microbial structures at high magnification
3. Certain staining protocols can help to differentiate between different types of micro-
organisms.
4. A stain (dye) is a salt composed of two ions, and one will contain the chromogen ( the
colored part of the molecule)
a. Positive ion (cation).
b. Negative ion(anion)
5. Most dyes used are basic dyes with a positive chromogen
 The interior of the cell ( cell’s cytoplasm) is negatively charged .
 The negative cytoplasm attracts the positively charged chromogen.
The ability of a stain to bind to macromolecular cellular components such as proteins or nucleic
acids depends on the electrical charge found on the chromogen portion as well as on the cellular
-component to be stained. Commonly there are two types of stains
1. Acidic stains (anionic) - Example: Picric Acid
2. Basic stains (cationic) - Example: Methylene Blue
| P a g e 27
Numerous staining techniques are available for visualization, differentiation and separation of
bacteria in terms of morphological characteristics and cellular structures. Broadly, staining
methods are simple staining and differential staining.
1. Simple staining is defined as the process of colouring bacteria or other cells by applying
a single solution of a stain to a fixed smear. It is used for visualization of morphological
shape (cocci, bacilli and spirilli) and arrangement (chains, clusters, pairs and tetrads) of
bacteria.
2. Differential staining is the use of more than one staining reagent to bring out differences
in microbial cell types, or to differentiate particular cellular components from the rest of
the cell body
| P a g e 28
All microbiological staining procedures require preparation of smears prior to the execution of
any of the specific staining techniques.
A. Preparation of smear: A good smear is one that, when dried, appears as a thin whitish
layer or film. Those made from broth cultures or cultures from a solid medium require
variations in technique.
1) Broth Cultures
One or two loops full of suspended cells should be applied directly to the glass slide
with a sterile inoculating loop and spread evenly over an area about the size of a
small coin.
2) Cultures from a solid medium

Organisms cultured in a solid medium produce thick, dense surface growth and are
not amenable to direct transfer to the glass slide. These cultures must be diluted by
placing a loopful of water on the slide in which the cells will be then emulsified.
Transfer of cells from the culture requires the use of a sterile inoculating needle. Only
the tip of the needle should touch the culture to prevent the transfer of too many cells.
Suspension is accomplished by spreading the cells in a circular motion in a drop of
water with the needle tip. The finished smear should occupy an area about the size of
a nickel and should appear as a semi-transparent, confluent, whitish film. At this
point, the smear must be allowed to dry completely. Avoidance of thick, dense smears
is absolutely essential.
NOTE: Students should use their own isolated bacteria to prepare this smear
| P a g e 29
B. Heat fixation
Unless fixed on the glass slide, the bacterial smear will wash away during the staining
procedure. This is avoided by heat fixation, during which the bacterial proteins are
coagulated and fixed to the glass surface. Heat fixation is performed by the rapid passage
of the air dried smear 2-3 times over the flame of the Bunsen burner.
Figure 12 : Bacterial Smear Preparation.
| P a g e 30
The use of a single stain to color a bacterial organism is commonly referred to as simple
staining. The purpose of simple staining is to elucidate the morphology and arrangement of
bacterial cells. Some of the most commonly used dyes for simple staining are methylene blue,
basic fuchsin, and crystal violet. All of these dyes work well on bacteria because they have
color-bearing ions (chromophores) that are positively charged (cationic). The fact that bacteria
are slightly negatively charged produces a pronounced attraction between these cationic
chromophores and the organism. Such dyes are classified as basic dyes (methylene blue ).
Those dyes that have anionic chromophores are called acidic dyes. Eosin is such a dye.
Exposure times differ for each of above stains: carbon fuchsin requires 15 to 30 seconds, crystal
violet 2 to 6 seconds, and methylene blue 1 to 2 minutes.
Material Required
Cultures: 24-hour nutrient agar slant cultures or nutrient broth of students’ isolated bacteria
Reagents: Methylene blue and crystal violet stain
Equipment:Bunsen burner,inoculating loop,staining tray, microscope, lens paper and glass slide.
A. Direct stain:
Procedure
1. Take clean glass slides, wash and dry them.
2. Prepare bacterial smears of both the cultures. All smears must be heat fixed prior to
staining.
3. Place the slide on the staining tray and flood the smear with one of the indicated
stains, using the appropriate exposure time.
4. Pour off the stain and wash smear with tap water to remove excess stain. During this
step, hold the slide parallel to the stream of water to reduce the loss of organisms
from the preparation.
5. Using blotting paper, blot dry the slide.
6. Examine the stained slides under oil-immersion.
| P a g e 31
Figure 13: Simple Staining Procedure.
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| P a g e 32
Figure 14 : Common Bacterial Shapes.
B. Indirect staining ( Negative Staining) :

Sometimes it is convenient to determine overall bacterial morphology without the use of harsh
staining or heat-fixing techniques that change the shape of cells. This might be the case when the
bacterium does not stain well (e.g., some of the spirochetes) or when it is desirable to confirm
observations made on the shape and size of bacteria observed in either a wet-mount or hanging
drop slide. Negative staining is also good for viewing capsules.
| P a g e 33
Negative, indirect, or background staining is achieved by mixing bacteria with an acidic stain
such as nigrosin, India ink, or eosin, and then spreading out the mixture on a slide to form a
film. The above stains will not penetrate and stain the bacterial cells due to repulsion between the
negative charge of the stains and the negatively charged bacterial wall. Instead, these stains
either produce a deposit around the bacteria or produce a dark background so that the bacteria
appear as unstained cells with a clear area around them .
Procedure
1. Use an inoculating loop to apply a small amount of bacteria to one end of a clean
microscope slide (figure 15 a).
2. Add 1 to 2 loops of nigrosin, India ink, or eosin solution to the bacteria (figure 15b)
and mix thoroughly.
3. Spread the mixture over the slide using a second slide. The second slide should be
held at a 45° angle so that the bacteria-nigrosin solution spreads across its edge
(figure 15c). The slide is then pushed across the surface of the first slide in order to
form a smear that is thick at one end and thin at the other (figure 15 d). This is known
as a thin smear.
4. Allow the smear to air dry (figure 15 e). Do not heat-fix!
5. With the low-power objective, find an area of the smear that is of the optimal
thickness for observation. Use the oil immersion lens to observe also.
Figure 15: Negative Staining Procedure and Thin Smear Preparation.
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Simple staining depends on the fact that bacteria differ chemically from their surroundings and
thus can be stained to contrast with their environment. Bacteria also differ from one another
chemically and physically and may react differently to a given staining procedure. This is the
principle of differential staining. Differential staining can distinguish between types of bacteria.
Gram stain
The Gram stain (named after Christian Gram, Danish scientist and physician, 1853–1938) is the
most useful and widely employed differential stain in bacteriology. It divides bacteria into two
groups—gram negative and gram positive.
The first step in the procedure involves staining with the basic dye crystal violet. This is the
primary stain. It is followed by treatment with an iodine solution, which functions as a
mordant; that is, it increases the interaction between the bacterial cell and the dye so that the
dye is more tightly bound or the cell is more strongly stained.
The smear is then decolorized by washing with an agent such as 95% ethanol or isopropanol-
acetone. Gram-positive bacteria retain the crystal violet-iodine complex when washed with the
decolorizer, whereas gram-negative bacteria lose their crystal violet-iodine complex and become
colorless.
Finally, the smear is counterstained with a basic dye, different in color than crystal violet. This
counterstain is usually safranin. The safranin will stain the colorless, gram-negative bacteria pink
but does not alter the dark purple color of the gram-positive bacteria. The end result is that gram-
positive bacteria are deep purple in color and gram-negative bacteria are pinkish to red in color.
How does Gram stain work?

differences in staining responses to the gram stain can be related to chemical and physical
differences in their cell walls. The gram-negative bacterial cell wall is thin, complex,
multilayered structure and contains relatively a high lipid content, in addition to protein and
mucopeptides. The higher amount of lipid is readily dissolved by alcohol, resulting in the
formation of large pores in the cell wall which do not close appreciably on dehydration of cell-
wall proteins, thus facilitating the leakage of crystal violetiodine (CV-I) complex and resulting in
| P a g e 35
the decolorization of the bacterium which later takes the counter stain and appears red. In
contrast, the gram-positive cell walls are thick and chemically simple, composed mainly of
protein and cross-linked mucopeptides. When treated with alcohol, it causes dehydration and
closure of cell wall pores, thereby not allowing the loss of (CV-I) complex and cells remain
purple When treated with a mordant Gram’s iodine, a crystal violet-iodine (CV-I) complex is
formed, resulting in deep purple colour in all cells.
Subsequent treatment with the decolorizer alcohol is the differentiating step. The alcohol quickly
removes the (CV-I) complex from the gram negative cells with ease whereas it takes a longer
time to be removed in gram positive cells. Thus, controlled treatment of alcohol for a limited
time period decolorizes all the gram-negative cells while the gram positive ones retain the purple
color. A counter stain such as safranine is then used to stain the colorless gram negative cells red.
The gram-staining procedure
Material
 Cultures: 18-24hour old agar culture or broth cultures of your isolated bacteria.
 Reagents: Crystal violet, Gram’s iodine, 95% ethyl alcohol and saffranine
Procedure
1. Flood smear with gram crystal violet primary stain and stain for 1 minute.
2. Wash off the crystal violet with cold water.
3. Flood the slide with Gram’s iodine mordant and let sit for 1 minute.
4. Wash off the mordant with safranin decolorizer/counterstain solution. Then add more
5. decolorizer/counterstain solution to the slide and stain for 20 to 50 seconds.
6. Wash off the decolorizer/counterstain with cold water.
7. Either blot or air dry
8. Observe under oil-immersion.
| P a g e 36
Figure 16 : The gram-staining procedure
| P a g e 37
Acid-Fast Staining (Ziehl-Neelsen method)

Most bacteria in the genus Mycobacterium contain considerable amounts of wax like lipoidal
material (mycolic acid), which affects their staining properties. Unlike most other bacteria, once
they are properly stained with carbolfuchsin, they resist decolorization with acidalcohol. Since
they are not easily decolorized they are said to be acid-fast. This property sets them apart from
many other bacteria.
This stain is used primarily in the identification of the tuberculosis bacillus, Mycobacterium
tuberculosis, and the leprosy organism, Mycobacterium leprae. After decolorization, methylene
blue is added to the organisms to counterstain any material that is not acid-fast; thus, a properly
stained slide of a mixture of acid-fast organisms, tissue cells, and non-acid-fast bacteria will
reveal red acid-fast rods with bluish tissue cells and bacteria.
The Ziehl-Neelsen acid-fast staining procedure (developed by Franz Ziehl, a German

bacteriologist, and Friedrich Neelsen, a German pathologist, in the late 1800s) is a very useful
differential staining technique that makes use of this difference in retention of carbolfuchsin.
Acid-fast microorganisms will retain this dye and appear red . Microorganisms that are not acid-
fast, termed non-acid-fast, will appear blue or brown due to the counterstaining with methylene
blue after they have been decolorized by the acid-alcohol.
Figure 17 : Ziehl-Neelsen acid-fast staining procedure
| P a g e 38
Some bacteria are capable of changing into dormant structures that are
metabolically inactive and do not grow or reproduce. Since these structures are formed inside the
cells, they are endospores. The German botanist Ferdinand Cohn discovered the existence of
endospores in bacteria. These are remarkably resistant to heat, radiation, chemicals and other
agents that are typically lethal to the organism. The heat resistance of pores has been linked to
their high content of calcium and dipicolinic acid. A single bacterium forms a single spore by a
process called sporulation.
The Sporulation takes place either by depletion of an essential nutrient or during unfavourable
environmental conditions. During sporulation, a vegetable cell gives rise to a new, intracellular
structure termed as endospore, that is surrounded by impermeable layers called spore coats.
Complete transformation of a vegetative cell into a sporangium and then into a spore requires 6
to 8 hours in most spore forming species. An endospore develops in a characteristic position
within a cell, i.e. either central, sub-terminal or terminal. Once an endospore is formed in a cell,
the cell wall disintegrates, releasing the endosperm that later becomes an independent spores.
Endospores can remain dormant for long periods of time. One record describes the isolation of
viable spores from a 3,000 year old archaeological specimen. However, a free spore may return
to its vegetative or growing state with the return of favorable conditions.
Endospores are formed by the members of several genera such as Bacillus, Clostridium, etc. The
spores are differentially stained by using special procedures that help dyes penetrate the spore
wall. An aqueous primary stain (malachite green) is applied and steamed to enhance penetration
of the impermeable spore coats. Once stained the endospores do not readily decolorize and
appear green within red cells.
| P a g e 39
Procedure
1. As described in previous exercises, aseptically transfer one species of bacterium with an
inoculating loop to each of the respective slides, air dry (or use a slide warmer), and heat-
fix.
2. Place the slide to be stained on a hot plate or boiling water bath equipped with a staining
loop or rack. Cover the smear with paper toweling that has been cut the same size as the
microscope slide.
3. Soak the paper with the malachite green staining solution. Gently heat on the hot plate
(just until the stain steams) for 5 to 6 minutes after the malachite green solution begins to
steam. Replace the malachite green solution as it evaporates so that the paper remains
saturated during heating . Do not allow the slide to become dry.
4. Remove the paper using forceps, allow the slide to cool, and rinse the slide with water for
30 seconds .
5. Counterstain with safranin for 60 to 90 seconds .
6. Rinse the slide with water for 30 seconds .
7. Blot dry with bibulous paper and examine under oil immersion. A coverslip is not
necessary. The spores, both endospores and free spores, stain green; vegetative cells
stain red.
Figure 18: Endospore Staining Procedure
| P a g e 40
| P a g e 41
Many studies require the quantitative determination of bacterial populations.

The two most widely used methods for determining bacterial numbers are the standard, or
viable, plate count method and spectrophotometric (turbidimetric) analysis. Although the
two methods are somewhat similar in the results they yield, there are distinct differences. For
example, the standard plate count method is an indirect measurement of cell density and reveals
information related only to live bacteria. The spectrophotometric analysis is based on turbidity
and indirectly measures all bacteria (cell biomass), dead or alive.
The standard plate count method consists of diluting a sample with sterile saline or phosphate
buffer diluent until the bacteria are dilute enough to count accurately. That is, the final plates in
the series should have between 25 and 250 colonies. Fewer than 25 colonies are not acceptable
for statistical reasons, and more than 250 colonies on a plate are likely to produce colonies too
close to each other to be distinguished as distinct colony-forming units (CFUs).
The assumption is that each viable bacterial cell is separate from all others and will develop into
a single discrete colony (CFU). Thus, the number of colonies should give the number of live
bacteria that can grow under the incubation conditions employed. A wide series of dilutions
(e.g., 10–4 to 10–10) is normally plated because the exact number of live bacteria in the sample
is usually unknown. Greater precision is achieved by plating duplicates or triplicates of each
dilution
(Benson 2002; Brown 2009; Freshney 2010; Harley 2004; Kayser 2005; Lacey 1997; Madigan et
al. 2012; Pollack 2004; Roberts et al. 2003; Steubing 1993; Tiwari 2009; Vandepitte et al. 2003)
| P a g e 42
Procedure: Standard Plate Count

1. With a wax pencil, label the bottom of five petri plates with the subsequent dilutions:10–2,
10–3, 10–4, 10–5and 10–6. Label five tubes of sterile distilled water 10–2, 10–3, 10–4, 10–
5
and 10–6
2. Using aseptic technique, the initial dilution of your isolated bacteria is made by
transferring 1.0 ml of liquid sample or 1 g of solid material to a 9-ml sterile distilled
water blank (figure 19). This is a 1/10 or 10-1 dilution. Cap the bottle.
3. The 10–1 blank is then shaken vigorously 25 times by placing one’s elbow on the bench
and moving the forearm rapidly in an arc from the bench surface and back. This serves to
distribute the bacteria and break up any clumps of bacteria that may be present.
4. Immediately after the 10–1 blank has been shaken, uncap it and aseptically transfer 1.0 ml
to a second 9ml sterile distilled water blank. Since this is a 10–1dilution, this second
blank represents a 10–2 dilution of the original sample. Cap the bottle.
5. Shake the 10–2 blank vigorously 25 times and transfer 1.0 ml to the third 9-ml blank. This
third blank represents a 10–3 dilution of the original sample. Cap the bottle. Repeat the
process once more to produce a 10–4 , 10–5and 10–6dilutions.
6. Shake the 10–2blank again and aseptically transfer 1.0 ml to one petri. Do the same for the
remaining blanks (figure 19).
7. After the pour plates have inoculated, they are inverted and incubated at 35°C for 24
hours or 20°C for 48 hours.
8. At the end of the incubation period, select all of the petri plates containing between 25
and 250 colonies. Plates with more than 250 colonies cannot be counted and are
designated too numerous to count (TNTC). Plates with fewer than 25 colonies are
designated too few to count (TFTC). Count the colonies on each plate. If at all possible,
a special counter such as a Quebec colony counter should be used (figure 19).
9. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the
number of colonies by the dilution factor . The number of colonies per ml reported should
reflect the precision of the method and should not include more than two significant
figures. For example, suppose the plate of the 10–6 dilution yielded a count of 130
colonies. Then, the number of bacteria in 1 ml of the original sample can be calculated as
follows: Bacteria/ml = (130) ÷ (10–6) = 1.3 × 108 or 130,000,000.
| P a g e 43
Figure 19 : Procedure Standard Plate Count
| P a g e 44
One method that is used to determine antibiotic susceptibility is the sensitivity disk method of
Kirby- Bauer (named after W. Kirby and A. W. Bauer in 1966). In this method, antibiotics are
impregnated onto paper disks and then placed on a seeded Mueller-Hinton agar plate using a
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 antibiotic. 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 a certain size indicates susceptibility,
zones of a smaller diameter or no zone at all show that the bacterium is resistant to the antibiotic.
Frequently one will see colonies within the zone of inhibition when the strain is antibiotic
resistant.
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
Figure 21: The Kirby-Bauer method
| P a g e 45
Procedure
First Period
1. With a wax pencil, mark the lid of each Mueller- Hinton agar plate with your name, and
date. Each student will inoculate the surface of Mueller-Hinton plate with his isolated
bacteria. Use a sterile cotton swab for inoculation. The swab is immersed in the culture
tube, and the excess culture is squeezed on the inner side of the test tube.
2. The swab is then taken and streaked on the surface of the Mueller-Hinton 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. Allow the
culture to dry on the plate for 5 to 10 minutes at room temperature with the top in place.
3. Dispense the antibiotics onto the plate either with the multiple dispenser or individually
with the single unit dispenser. Make sure that contact is made between the antibiotic disk
and the culture by gently pressing the disk with alcohol-flamed forceps.
4. Incubate the plates for 16 to 18 hours at 35°C. DO NOT INVERT THE PLATES.
Second Period:
1. Measure the zones of inhibition to the nearest mm for each of the antibiotics tested.
Figure 21 : The antimicrobial susceptibility disk diffusion test: approximate disk

placement and measurement of inhibition zone diameters.ATB1 = antibiotic 1, ATB2
= antibiotic 2, etc
| P a g e 46
REFERENCES
1. Benson HJ. 2002. Microbiological applications: a laboratory manual in general
microbiology: McGraw-Hill New York, NY.
2. Brown AE. 2009. Benson's Microbiological Applications: Laboratory Manual in General

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3. Freshney RI. 2010. Culture of animal cells: a manual of basic technique and specialized
applications: Wiley-Blackwell.
4. Harley JP. 2004. Laboratory exercises in microbiology: McGraw-Hill

Science/Engineering/Math.
5. Kayser FH. 2005. Medical microbiology: Georg Thieme Verlag.
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Laboratory Manual in General Microbiology For Undergraduate Students., Short

Book · September 2012

Sulaiman M Alnaimat Saqr Abushattal

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[Laboratory Manual in General Microbiology]

[Laboratory Manual in General Microbiology]

Some Microbiological Equipments ................................................................................................ 3

Microbiology Lab Practices and Safety Rules ................................................................................ 4

The Control of Microbial Growth ................................................................................................... 5

Microbiological Culture Media Preparation .................................................................................. 7

Isolation of Microorganisms from anEnvironment of Choice ...................................................... 10

Culture Transfer Instruments, Techniques, and Isolation of Pure Cultures .................................. 12

Microscopic Techniques ............................................................................................................... 18

Bacterial Morphology and Staining .............................................................................................. 27

Preparation of smear ..................................................................................................................... 29

Simple Staining (direct and indirect staining) ............................................................................. 31

Differential stain ( Gram Stain & Acid Fast Stain)....................................................................... 35

Structure stain ( Endospores stain) ........................................................................................... 39

Determination of Bacterial Numbers ............................................................................................ 42

Effects of Chemical Agents on Bacteria: ...................................................................................... 45

Bunsen burner Laminar air flow Compound Microscope

Water bath Autoclave Refrigerator

Incubator Balances Magnetic stirrer

Sterilization is the complete destruction or elimination of all viable organisms (in or on an

Heat: most important and widely used..

4. Pasteurization : s a process of heating a food, which is usually a liquid, to a specific

Antiseptics: Microbicidal agents

Disinfectants: Agents that kill

Chemical and gas:

Irradiation (microwave, UV, x-ray, Gamma radiation): destroys microorganisms as described

Nutrition materials in media :

Agar : is a gelatinous polymer substance derived

Figure2 : The different types of Solid media

Classes of Culture Media ( Composition)

2. Complex media (undefined media) employ digests of microbial, animal or plant

3. An enriched medium, often used for the culture of otherwise difficult-to-grow

Classes of Culture Media ( Purpose)

 Preparing a broth Medium

 Preparing a Agar Medium

 Procedure for Autoclaving

Aseptic technique : refers to a procedure that is performed under sterile conditions.

 Procedure for Pouring autoclaved culture media

Figure 3 : pouring a plate

1. Place 1 g of garden soil in a 99-ml sterile water blank

Figure4 : Enumerating soil microorganisms

For isolation bacteria from other sources

Suggested procedure to use to achieve aseptic technique

Contamination Risk Precaution

Microbiological Transfer Instruments

1. Inoculating loops and needles:

Figure 6 : Good flaming technique. Flaming begins at the handle and

Techniques for isolation of pure culture:

1. The streak –plate method:

Pure culture: laboratory

2. Pour plate method

3. Spread plate method

Essential features of various microscopes