MICROBIOLOGY LABORATORY SAFETY 7.

Laboratory coats and other protective

RULES AND REGULATIONS wear should stay inside the laboratory and
should not be worn outside (i.e. when
All students enrolled to the microbiology obtaining needed materials in the
course are required to read and assimilate stockroom or when operating the
the information in this document with autoclave)
regard to laboratory safety prior to the 8. Do not eat, drink, smoke or apply
first laboratory session of each academic cosmetics inside the laboratory.
term. Students must also adhere to 9. Do not engage in rowdy, playful, or
written and verbal instructions throughout unprofessional activities (such as watching
the semester. Additional instructions are videos and playing mobile games) in the
usually given at the beginning of each laboratory.
laboratory class and for this reason, it is 10. Do not leave an on-going experiment
important that all students arrive at each or activity unattended (especially while
session on time. operating the autoclave and cooking
media in the microwave oven).
PERSONAL SAFETY GUIDELINES 11. Do not work alone in the laboratory.
1. Long nails should be trimmed short 12. Never do any unauthorized
before coming to the laboratory class. Nail experiment or activity.
polish and artificial nails should also be
removed. GENERAL LABORATORY ETIQUETTE
2. Long hair should be securely tied 1. Read carefully all the labels inside the
behind the head. A cloth cap could be laboratory and do not displace any
worn to contain the hair. materials that are to be found in the
3. Cell phones must be deposited before working benches.
the start of each class. A specific box will 2. Label clearly all materials with its name,
be provided to keep the cell phones. Cell date, and any other applicable
phones will only be allowed when needed information.
(such as to take pictures of your samples, 3. Working benches are to be kept free of
set-ups, etc.). extraneous items while conducting
4. Bags will be left outside the classroom experiments. This includes personal items
throughout the duration of the class. such as backpacks, mobile phones, laptops
Laboratory materials and needed items and unnecessary books.
(such as laboratory exercise, ballpens, 4. The surfaces of the working benches
notebook) must be brought in the must be disinfected at the beginning and
laboratory room before the start of the at the end of every laboratory period by
class. using 70% alcohol or 10% bleach.
5. Personal accessories (watches) and 5. Do not take the consumable materials
pieces of jewelry (rings, dangling earrings of other groups without permission and
and necklaces, bracelets) are required to replace what you have used.
be removed before going inside the 6. Do not pour bio hazardous fluids down
laboratory. the sink. Autoclave liquids and broth
6. No one is allowed to enter the cultures to decontaminate them before
laboratory without their closed-toe discarding.
laboratory shoes, buttoned long-sleeved 7. Chemicals must be disposed of properly.
laboratory coats, laboratory gloves, Special containers are designated for
laboratory masks and safety glasses.
some chemicals for disposal to prevent 8. Do not leave a heat source (alcohol
environmental contamination. lamp, hot plate or Bunsen burner)
8. Trash must be disposed properly. Items unattended. Keep containers of alcohol,
contaminated with microorganisms must acetone, or other flammable liquids at a
be autoclaved before disposal or washing. safe distance from flames.
Solid waste materials must be placed in a 9. Keep hands away from your face, eyes,
biohazard bag and be autoclaved before and mouth when working with chemicals
disposal. or microorganisms. This includes not
9. After each session, return all chemicals, applying cosmetics, not adjusting contact
reagents, media and cultures to their lenses, and not biting your finger nails.
appropriate places. 10. Never pipette by mouth. Use pipette
10. The laboratory must be clean including bulbs or pipetting devices for the
the floor before leaving. Laboratory stools aspiration and dispensing of liquid
must be kept under the tables. cultures.
11. Check glassware for cracks and chips
GOOD MICROBIOLOGICAL LABORATORY before using as it could eventually cause
PRACTICES the glassware to fail during use and may
1. Keep doors and windows closed during cause serious injury. Always inform the
each laboratory session to prevent instructor when a glassware has cracks,
contamination from air currents. chips or is broken.
2. Students must wash their hands
thoroughly (using soap and water) before MATERIALS AND EQUIPMENT ETIQUETTE
and after each laboratory activity and 1. Glassware and other materials used in
before leaving the laboratory. each laboratory session should be washed
3. Treat all microorganisms—especially properly with liquid detergent
unknown cultures—as if they were (disinfectant) completely removing culture
pathogenic. A student who had been media residues, chemicals, and biological
recently ill or has a compromised immune samples.
system should inform/consult the 2. Any materials spilled on the balance
instructor before working in the must be cleaned/removed thoroughly.
microbiology laboratory. 3. Microscope must always be wiped clean
4. Practice Aseptic Technique. It helps before putting it away and must be in
ensure that only one type of proper posture after using.
microorganism is present in a pure 4. Biological safety cabinet must be kept
culture. This also ensures that the clean and should not be used as storage of
microorganism does not escape from the media, culture plates and other materials.
container, contaminating the laboratory, 5. Microbiological incubator must be kept
and possible causing disease. clean and in an orderly state.
5. All materials needed for the execution 6. Refrigerators are used to store
of the experiment must be sterilized. microbiological samples and cultures. Do
6. Inoculating loops and needles should be not store any unnecessary material (i.e.
flame sterilized before and after using. food, drinks, anything other than what will
7. Take special precaution when working be used in the microbiology laboratory)
with open flames. Loose hair, clothing, 7. The microwave oven is intended for
dangling jewelry, ID lanyards and nearby media preparation only and should not be
paper must be secured. used for any other purpose (i.e. heating
food and drinks). It should be kept clean at
all times – any spill or stain must be controls the amount of light coming
removed after using. through it. Both coarse and fine
8. Autoclave must be cleaned before and adjustments are found on the light
after use, and replacing the water to microscope.
completely remove residues of To magnify an object, light is projected
microbiological culture media that may through an opening in the stage, where it
promote unwanted growth of bacteria hits the object and then enters the
(leading to foul odor) when the unit is objective. An image is created, and this
stored for a longer time. image becomes an object for the ocular
9. Container of culture media and lens, which remagnifies the image. Thus,
chemicals should also be checked and be the total magnification possible with the
properly sealed after using to maintain the microscope is the magnification achieved
quality or shelf-life. by the objective multiplied by the
10. All electrical equipment that are not in magnification achieved by the ocular lens.
use shall be unplugged from wall sockets
before leaving the laboratory. A compound light microscope often
contains four objective lenses: the
EXERCISE NO. 1 Microscopy scanning lens (4X), the low‐power lens
I. Introduction (10X), the high‐power lens (40 X), and the
The microscope is one of the basic tools oil‐immersion lens (100 X). With an ocular
used in the conduct of both basic and lens that magnifies 10 times, the total
advance biological research. Although magnifications possible will be 40 X with
students are acquainted with microscopes the scanning lens, 100 X with the low‐
early in their biological science classes, power lens, 400 X with the high‐power
not all students learn how to use and care lens, and 1000 X with the oil‐immersion
for microscopes. In many cases, students lens. Most microscopes are parfocal. This
are grouped in the laboratory sessions and term means that the microscope remains
not every student of each group would in focus when one switches from one
take the opportunity to learn how to set objective to the next objective.
up the microscope. This activity allows
each student to gain essential microscopy The ability to see clearly two items as
skills including the calibration of the separate objects under the microscope is
microscope. It also provides a review on called the resolution of the microscope.
the basic workings of a compound light The resolution is determined in part by
microscope. the wavelength of the light used for
observing. Visible light has a wavelength
The common light microscope used in the of about 550 nm, while ultraviolet light
laboratory is called a compound has a wavelength of about 400 nm or less.
microscope because it contains two types The resolution of a microscope increases
of lenses that function to magnify an as the wavelength decreases, so
object. The lens closest to the eye is called ultraviolet light allows one to detect
the ocular, while the lens closest to the objects not seen with visible light. The
object is called the objective. Most resolving power of a lens refers to the size
microscopes have on their base an of the smallest object that can be seen
apparatus called a condenser, which with that lens. The resolving power is
condenses light rays to a strong beam. A based on the wavelength of the light used
diaphragm located on the condenser and the numerical aperture of the lens.
The numerical aperture (NA) refers to the
widest cone of light that can enter the II. Learning Objectives
lens; the NA is engraved on the side of the At the end of this exercise, students will
objective lens. be able to:
If the user is to see objects clearly, 1. have a review on the proper care and
sufficient light must enter the objective maintenance of microscopes.
lens. With modern microscopes, entry to 2. learn the steps for proper focusing on
the objective is not a problem for material and observation with a
scanning, low‐power, and high‐power microscope.
lenses. However, the oil‐immersion lens is 3. learn how to determine the
exceedingly narrow, and most light misses magnification of materials being observed
it. Therefore, the object is seen poorly and in a microscope.
without resolution. To increase the 4. learn how to determine size of
resolution with the oil‐immersion lens, a specimen being observed in a microscope
drop of immersion oil is placed between
the lens and the glass slide. Immersion oil III. Materials
has the same light‐bending ability (index compound light microscopes with oil
of refraction) as the glass slide, so it keeps immersion objective
light in a straight line as it passes through ocular/ eyepiece micrometers
the glass slide to the oil and on to the stage micrometers
glass of the objective, the oil‐immersion immersion oil
lens. With the increased amount of light prepared slides of bacteria, fungi, algae
entering the objective, the resolution of and protist
the object increases, and one can observe
objects as small as bacteria. Resolution is IV. Procedure
important in other types of microscopy as Calibrating the Microscope
well. To properly calibrate your eyepiece with a
stage micrometer using the low power
Care and Cleaning of the Compound objective (LPO), align the zero line
Binocular Microscope (beginning) of the stage micrometer with
Since all microscopes are delicate the zero line (beginning) of the eyepiece
precision instruments they must be well micrometer (Figure 1). Carefully scan over
cared for. It is important to lift them from until you see the lines line up again.
the cabinet with the use of two hands. Determine the value that each line
One hand should grab the microscope by represents in your eyepiece.
its arm and the other hand should be
place under the base of the microscope to
ensure the microscope is firmly held. Since
the microscope you will be using is also
used by several other students it is
important to clean the ocular lens and the
objective lens each lab period before and
after use. To do this take a piece of lens
paper and dampen it with the lens In the example above, the stage
cleaning solution. It is important to only micrometer is 1 mm long with 100
use lens paper and not paper towels, since divisions so each division of the stage
they will scratch the lens. micrometer is one one-hundredth of a
mm (0.01mm or 10 um). The eyepiece The survival and growth of
micrometer is divided into 100 units. We microorganisms depend on available and a
don't need to know the actual distance favorable growth environment. Culture
between the lines on it. When the zero media are the nutrient solutions used in
marks are lined up, scan across and look laboratories to grow microorganisms. For
for a convenient point where the lines the successful culture of a given
converge again. In Fig.1, the 45 mark on microorganism it is necessary to
the eyepiece micrometer is closely in line understand its nutritional requirements
with the stage micrometer. Count the and then supply it with its essential
divisions comprising the aligned divisions. nutrients in the proper form and
The measurement that each line in the proportions in a culture medium. The
eyepiece represents can now be general composition of a medium is as
determined. This will be considered as follows:
your calibration factor, which can be 1. H-donors and acceptors (approximately
computed using the following formula: 1-15 g/L)
2. C-source (approximately 1-20 g/L)
3. N-source (approximately 0,2-2 g/L)
4. Inorganic nutrients e.g. S, P, (50mg/L)
5. Trace elements (0,1-1 µg/L)
Microscopic observation of cells 6. Growth factors (aminoacids, purines,
1. Keep the ocular/ eyepiece micrometers pyrimidines, occasionally 50 mg/L,
in place. Observe the different prepared vitamins occasionally 0,1-1 mg/L)
slides using the LPO. For bigger specimen, 7. Solidifying agent (e.g agar 15g/L)
the scanner may be used and for smaller 8. Solvent (usually distilled water)
ones, the high power objective (HPO) may 9. Buffers
be used. However, the objectives should
be calibrated first. According to the consistency three types
2. Using the eyepiece micrometer and of media are used: liquid, or broth, media;
with knowledge on the calibration factor, semisolid media; and solid media. The
measure the length of the cells and the major difference among them is that solid
distinct organelles that can be observed. and semisolid media contain a solidifying
(Note: due to the availability of the or gelling agent [such as agar, gelatine],
eyepiece micrometers, some may whereas a liquid medium does not.
estimate the cell measurements using the • Liquid media, such as nutrient broth,
computed FOV). tryptic soy broth or glucose broth can be
3. Document, label and indicate the used in studies of growth and metabolism
measurements. Size is best indicated by in which it is necessary to have
including a ruler bar at the bottom of the homogenous media conditions, to follow
drawing, drawn to scale and with the optical density, and to allow early
dimensions added. Place a line (bar) to sampling for analysis of substrates and
indicate a length of 10 microns. Include metabolic products. Tubes and flasks with
the magnification used to view each. liquid cultures can be incubated with
either static or shaken incubation.
EXERCISE NO. 2 CULTURE MEDIA • Semisolid media can also be used in
PREPARATION AND STERILIZATION fermentation studies, in determining
I. Introduction bacterial motility, and in promoting
anaerobic growth.
• Solid media, such as nutrient agar, are growth of others due to the inclusion of
used particular substrate. Differential media
1) for the surface growth of allow identification of microorganisms
microorganisms in order to observe colony usually through the (visible) physiological
morphology, reactions unique to those bacteria. The
2) for pure culture isolation, most practical media are those that both
3) often in the enumeration and isolation select for and differentiate common
of bacteria from a mixed population by pathogens. Enrichment media allow
diluting the original bacteria suspension metabolically fastidious microorganisms to
and spreading a small inoculum over the grow because of the addition of specific
surface of the solidified medium and growth factors. Enrichment culture is one
4) to observe specific biochemical obtained with the use of selected media
reactions (extracellular enzymes diffusing and incubation conditions to isolate the
away from the colony can be detected as a desired microorganisms from natural
result of their action on insoluble samples.
substrates present in the agar medium).
Solid media can be poured into either a II. Learning Objectives
test tube or Petri dish. If the medium in At the end of this exercise, students will
the test tube is allowed to harden in a be able to:
slanted position, the tube is designated an 1. acquire skill in culture media
agar slant; if the tube is allowed to harden preparation 2. determine the steps in
in an upright position, the tube is sterilization of culture media and
designated an agar deep tube; and if the equipment
agar is poured into a Petri dish, the plate is 3. apply aseptic technique in pouring
designated an agar plate. culture media on petri plates

Media are categorized by their III. Materials

composition: chemically defined, or Alcohol Lamp, Erlenmeyer’s Flask, Petri
synthetic, media are composed of known Plates, Cotton Plugs, Gelatin (for practice
quantity and quality of pure chemicals. In of pouring agar), Autoclave with Regulator,
routine bacteriology laboratory exercises, Electric Stove
complex or nonsynthetic media are
employed. These are composed of IV. Procedure
complex materials rich in vitamins and Culture Media Preparation
nutrients, the chemical composition of The preparation of media from
which is poorly defined. Three of the most commercially available dehydrated
commonly used components are beef products is simple and straightforward.
extract, yeast extract and peptone Each bottle of dehydrated medium has
(partially digested protein). instructions for preparation of its label.
For example, to prepare a liter of tryptic
Media can also be categorized by their soy broth, suspend 30 g of the dehydrated
function: An all-purpose medium, such as medium in 1000 ml distilled water. Mix
Tryptic Soy Agar, supports the growth of thoroughly in a 2 liter Erlenmeyer flask
most bacteria cultured in the laboratory. [always use a flask that holds twice the
They do not contain any special additives. volume of media you are preparing].
Selective media enhance the growth of Dispense and sterilize for 20 minutes at
certain organisms while inhibiting the 121°C [15 lbs pressure]. As noted, the
amount of powder for 1000 ml of water of the air has been expelled and only
will be indicated. In case of preparation of steam is present in the autoclave chamber.
media from a formula, pour 500 ml of They are carefully temperature controlled
distilled water into a 2000ml Erlenmeyer as well. Almost all media and anything
flask. Then measure adequate amount of else that will resist 121°C and steam can
media components and dissolve be sterilized this way. Often dry glassware,
completely in the water in the order of the such as pipettes and Petri plates, must be
formula. At the end rinse the flask with sterilized. Steam tends to etch glassware
the remaining 500 ml water. Mix the and also leaves it damp. Therefore, such
medium thoroughly, adjust the pH and items are generally dry – heat sterilized.
sterilize. If the medium lacks agar, the The glassware is placed in an electric oven
powder will usually dissolve without set to operate between 160 and 170°C.
heating. If it contains agar, it is necessary Since dry heat is not as effective as wet
to heat the medium until it starts to boil or heat, glassware must be kept at this
even longer in order to completely temperature for about 2 hours or longer.
dissolve the agar. Most of the exercises Oven temperature must not rise above
you will be doing will involve the use of 180°C or any cotton or paper present will
sterile media culture tubes. These tubes char. Sometimes media must be made
must be capped in order to maintain from components that will not withstand
media sterility. This can be accomplished heating at 121°C. Such media can be
by using cotton plugs, plastic foam plugs sterilized by passing it through a
or plastic or metal caps. All of these caps (bacteriological) filter, which physically
keep cultures free of contamination while removes bacteria and larger
allowing air into the culture tube, and microorganisms from the solution and
minimizing evaporation at the same time. thereby sterilizes them without heat.
It is sometimes desirable to use screw cap Scintered glass filters with ultrafine fitted
culture tubes. This is especially true when disks [0.9 to 1.4 µm pore size] and Seitz
the culture, such as in the case of slants, asbestos - pad filter funnels [3 mm thick
may be sealed and stored for long periods. with 0.1 µm pores] are both quite
Culture broth can be dispensed with the effective sterilizing solutions. The most
pipetting machine, an automatic syringe, useful and popular approach by far is the
or a regular pipette. use of specially prepared sterile, cellulose-
or polycarbonate, etc.- based membranes
Sterilization of Media and Equipment of the appropriate pore size. Generally,
Sterilization is the process of rendering a membranes with 0.22 µm pores are
medium or material free of all forms of employed in sterilization. A large number
life. There are three basic ways in which of different devices are commercially
sterilization of media and supplies can be available for membrane sterilization of
achieved. The most useful approach is both large and small volumes. For
autoclaving, in which items are sterilized example, one can use a filter flask with
by exposure to steam at 121°C and 15 for vacuum or syringe with positive pressure
15 minutes or longer, depending on the to force liquid through a special
nature of the item. Under these membrane filter holder.
conditions, microorganisms, even
endospores, will not survive longer than EXERCISE NO. 3 BASIC MICROBIOLOGICAL
about 12 to 13 minutes. Modern TECHNIQUES
autoclaves are designed to ensure that all I. Introduction
An experienced microbiologist employs 2. determine the steps in performing the
many techniques and skills when handling streak plate technique
microorganisms. More so than in any
other field of science, the skills of a III. Materials
microbiologist need to be practiced and Alcohol Lamp, Inoculating Loop, Agar
refined to achieve a level of proficiency. Slant, Agar Plate
Microscopy is one skill you are already
learning and will soon master with further IV. Procedure
practice. Other skills of the microbiologist A. Practice Aseptic Technique
allow for the safe handling and The technique that you will use most
manipulation of microorganisms. Isolation frequently during the semester is aseptic
and investigation of microorganisms are transfer of bacteria, which you will do
formidable tasks confronting both novice whenever you inoculate culture media
and experienced microbiologists. The skills with an organism. The inoculating loop
employed to achieve these tasks are called (shown to the right) is the standard tool of
aseptic technique. the microbiologist and is used to transfer
Aseptic technique reduced the potential cells to and from culture media. An
spread of bacteria used in laboratory to important point to remember is that
you or other people. This could happen if seeing a mass of bacteria on the loop
a pathogen were allowed to escape the during a transfer is not necessary
lab on contaminated clothing, books, or (although it is reassuring) even a single cell
other materials. You will be introduced is adequate to start a new culture. An
this week to the basic skills of aseptic essential rule for any aseptic transfer is to
technique, and will be expected to apply sterilize the inoculating loop BEFORE and
and refine them over the course of the AFTER each transfer. Why? The techniques
semester. Aseptic technique is necessary for doing aseptic transfers will be
to prevent contamination of pure cultures demonstrated in class. The manner in
(a culture containing a single species) which you hold the tubes, caps and
which are used routinely for analysis. inoculating loop are important. You are
Extraordinary precautions are needed to encouraged to ask for assistance or a
prevent contamination because critique while you perform the transfers.
microorganisms, particularly bacteria, are
‘everywhere’. Trillions of bacteria occur in, You will be given opportunity during the
on and around our bodies, and even slight lab period to learn the proper techniques
carelessness can lead to inadvertent for holding tubes, plates and the
contamination. The vast majority of these inoculating loop so that you learn how to
bacteria are harmless, even beneficial, but make transfers autonomously (i.e.,
analysis of a contaminated culture will without someone else also holding the
yield unpredictable and most likely tubes)
erroneous results
Each student should:
II. Learning Objectives
1. Transfer microorganism from a plate to
At the end of this exercise, students will
an agar slant and vice-versa
be able to:
2. Transfer microorganism from an agar
1. acquire skill in transferring inoculum
slant to another agar slant.
from a culture while applying aseptic
3. Transfer microorganism from a broth
techniques
culture to an agar slant
4. Incubate all of the inoculated cultures
at 37°C for 24 - 48 hours, and then
observe whether the transfers were
successful, and then discard the cultures.

B. The Streak Plate Technique

Streak plating is a powerful technique that
microbiologists employ for a number of
purposes, including 1) evaluating the
purity of a bacterial culture, 2) examining
the diversity of species in a sample, 3)
separating a species from a mixed culture
so that a pure culture can be prepared,
and 4) studying the colony characteristics
of a species. The streaking technique will
be demonstrated during the lab period;
however, the general procedure and
strategy is described below. Most
commonly streak plating is carried out to
obtain isolated colonies. Remember, a
"colony" is bacterial growth on solid
medium that originated from a single cell.
To obtain isolated colonies, individual cells
must be dispersed over the surface of the
agar medium. This is accomplished by
spreading the sample as a series of
"streaks" on solid medium in a petri plate,
as shown in Figure 1. The pattern of
streaking shown below consistently yields
good results in this course, but is not the
only technique that can be used. Note
that the inoculating loop is flame sterilized
between each step, and with each series
of streaks the density of cells is decreased.