Laboratory safety for 3rd year medicine students for Clinical Microbiology. Microscopy and Bacterial Morphology

1. Microbiology practical
3nd year medicine
Clinical Microbiology
1st Principles of Bacteriology
OBJECTIVES
1. To know and understand the principle of bacteriological identification
tests .
2. To know what specimen to take from a patient and what test (s) to
request from the bacteriology laboratory.
3. To be able to interpret the results of laboratory tests to aid in the
final diagnosis.
UQU Micro. Raniah 1429-1430 1

2. BASIC L
LABORAT
TORY SA
AFETY M
MEASUR
RES
You m be stric adhere to it, an so you can protect yourself, your colle
must ctly ed nd c t eagues and
d
your famiily.
FOLL
LOWING SSAFETY M
MEASURE CONSI
ES IDERED I YOU LAB EVAL
IN LUTION.
1. We a full length zip
ear pped up w
white lab coat.
2. We gloves when working.
ear
3. No open sho allowe
o oes ed.
4. Lo hair must be tied back and da
ong k, angling je
ewelry an baggy
nd y
clo
othing mu be secu
ust ured.
5. No eating, s
o smoking o drinkin in the lab.
or ng
6. Do not put a
o mouth e.g. pencils, p
anything in your m pipettes, f
fingers, lip balm.
p
7. No personal belongin allowe to the lab (e.g.; bags).
o l ngs ed l
8. Be
efore leavi the la remov your co hang it or put it in a separ bag. D
ing ab, ve oat t rate Do
not wear you work la coat out
t ur ab tside the departmen
d nt.
9. Keeep your area o work tidy put away ite you are finish
r of t ems hed wi
ith,
dis
scard wast material you have finished with.
te l e
10. De
econtaminate your area with the prov
h vided disi
infectant (
(Dettol co
oloroxylen
nol
50% 20%), or hypoch
%- hlorite.
11. Sm spilla must b moppe up with disinfect
mall age be ed h tant.
efore and after the lab time).
12. Wa your hands (be
ash d e
ACCIDE ENTS AN INJUR
ND RIES:
13. Report any a
accident (sp breakage, etc.) or injury (c burn, etc.) to the superviso
pill, o cut, e or
imm
mediately..
14. Tre minor c and ab
eat cuts brasions a follows:
as
a) Wa thorou
ash ughly.
b) Alllow to ble freely.
eed
c) Dr and apply elastopl
ry last.
d) En details in acciden book an notify th lab seni
nter s nt nd he ior.
15. If y or you lab partn is hurt, immediat
you ur ner , tely yell o the sup
out pervisor's. Do not
pannic.
16. . If a chemical should splash in y
f your eye(s or on yo skin, im
s) our mmediatel flush wi
ly ith
runnning wat for at l
ter least 20 m
minutes.
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icro. Raniah 1429-1430
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3. 1.INTRODUCTION TO LABORATORY SAFETY
1. OBJECTIVES:
• To get familiar with general safety measures considered necessary to work I n a
microbiology laboratory, and when dealing with basic medical (biohazard) waste.
• To make awareness of how important is the use of safety cabinet in the handling
of infectious material, in particular microorganism.
2. BACKGROUND
a wide variety of specimen are received daily in a microbiology laboratory, many of
them containing pathogenic microorganisms. Laboratory acquired infections have
always been a hazard for those working in the medical laboratories and similar
environment. Infection in microbiology laboratories is generally caused by
inhalation, inoculation, or ingestion of microorganisms. Release of
microorganisms in the form of aerosols increase the risk of infection by inhalation
unless the aerosols are contained or restricted. Aerosols can be created; by culturing
microorganisms, particularly from fluid specimens; by accidents, such as breakage
in a centrifuge; or by the dropping of culture. To contain aerosols, “safety
cabinet” should be used for all manipulations likely to produce
infected aerosols.
ROUTS OF ENTRY: (HOW WE GET INFECTED)
Infection in microbiology laboratories is generally caused by
1. Inhalation (through the lung) inhalation of airborne microorganisms
2. Inoculation (conjunctiva, skin injuries) skin - injuries by needles, sharp
instruments, or glass. Animal bites and scratches. Cuts and scratches.
conjunctiva - splashes of infectious material into the eye, transfer of
microorgansims to eyes by contaminated fingers
3. Ingestion (mouth) through eating, drinking, and smoking in the
laboratory, mouth pipetting, transfer of microorganisms to mouth by
contaminated fingers or articles.
HAND WASHING Hand washing in right way and right time is basic and
important procedure in protecting the laboratory worker as well as the patient and
the hole community form getting infected with so many dangerous infectious
diseases. Especially those microbes which can be easily transmitted via direct
contact. Figure (1).
Safety is the responsibility of every member of the laboratory including the head of
the department. Although safety on laboratories relies predominantly on the common
sense of individuals, it is necessary to lay down general guidelines and rules, which
must be in practice at all times. One such guidelines is prepared and attached here
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4. for all the students and supervisors to follow while they are working in the
laboratory(page 2 ).
Clean Hands
Save Lives.
Note: Rubbing with soap (step 1 t0 6) should be done for 20 seconds. And repeated if needed
tell the hand is clean
Figure ( 1 ): Hand Washing.
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5. 2. MEDICAL WASTES
BLACK Or TRANSPARENT BAGS
• PAPERS , WATER BOTTLES…etc
(basket OUTSIDE THE LAB)
A. CONTAMINATED MATERIALS
• GLOVES Yellow Bags with biohazard sign.
• CONTAMINATED PLASTIC LOOPS
Discard Jars with disinfectant.
• CONTAMINATED WIRE LOOP
Red hot (flaming)
• CULTURE PLATES WITH GROWTH
Yellow or Red square basket.
• INFECTED SHARPS (Syringes' Needles, Glass Slides)
Sharp container clearly
labeled (NSI).
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6. 3. BIOLOGICAL SAFETY CABINETS
The biological safety cabinets can be of three types:
1. Class I safety cabinets:
Class I safety cabinet Figure( 2), have an open front with negative pressure
ventilation and a HEPA- filtered air exhaust system. These cabinets are designed
to protect the user from infectious agent. Class I cabinets are not suitable for cell
culture operations .
HEPA
filter
Glass
panel
(decontaminated air)
Figure (2): biological safety cabinets class I
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7. 2. The Class II safety cabinets:
a. These are the best all-round biological safety cabinets for HEPA filter
general microbiological usage. Figure (3).
HEPA
filter
Glass panel
(decontaminated air)
Figure (3): biological safety cabinets class II
b. class II cabinets provide protection to the user, the environment, and the
culture by means of a recirculating HEPA-filtered vertical airflow, and
HEPA-filtered exhaust air.
3. The Class III cabinets:
Ventilated cabinet - totally enclosed. It provides both personnel and specimen
protection .Operations are
conducted through attached HEPA filter
rubber gloves. Both supply HEPA
and exhaust air are HEPA- filter
filtered. Glass
Gloves
(decontaminated air)
Figure (4): biological safety cabinets class III
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8. Practical No 2
LIGHT MICROSCOPE
1. OBJECTIVES:
1.1. To be familiar with different parts of a compound microscope.
1.2. To be familiar with the major application of light microscope.
1.3. To know the use of each objectives lenses.10X for wet preparation, and 100X
for oil immersion. To adjust the light source to optimum depending on the
preparation being examined by using the condenser and iris diaphragm.
2. BACKGROUND
The use of microscope in all their various forms in known as microscopy. the
microscope is used in the microbiology laboratory to study microorganism. Using a
system of lenses and illumination sources, it makes a microscopic object visible.
Microscopes can magnify an abject from 100-100 times of its original size. The size of
bacteria are always expressed on metric units such as millimeter (mm), micrometer
(µm) and nanometer (nm). (1 mm= 1000 µm or 1000,000 nm). Figure (5).
Figure ( 5): Range of sizes of major microorganisms, and the range of human eye,
light microscope, and electron microscope.
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9. 2.1. Light microscope
Principle: to magnify an abject the light microscope uses a system of lenses (objectives
and oculars) to manage the path of light beam that travels between the object being
studied and the eye.
2.2. Application of the light microscope
2.2.1.Bright field microscopy
It uses a light source that illuminate the entire specimen field. This method is used to
examine stained preparation and sometimes non-stained.
2.2.2.Dark field microscopy
It uses light microscope equipped with a special condenser and objective to brightly
illuminate the microorganism in the specimen against a dark background. This method
is used for the examination of unstained motile living microorganisms e.g. Treponema
species.
2.2.3.Fluorescent microscopy
Ultraviolet lamp is used instead of ordinary light bulb. The specimen is stained with
fluorescent dye that absorbs the energy of short light waves (ultraviolet). The dye then
released or emits light of long wavelength such as green light (fluorescence).
Commonly used fluorescent dyes are acridine orange, auramine/ rhodamine, and
calcoflour white.
2.2.4. Phase contrast microscopy
It uses a modified light microscope that permits greater contrast between substances of
different thickness or density. A special condenser and objective controls the
illumination. The result is an image of structure with differing degrees of brightness or
darkness, collectively called contrast. The denser materials appear bright and the part
of cell that have a density close to water will appear dark.
3. MATERIALS
1. Compound microscope.
2. Lens cleaning paper/cloth.
3. Immersion oil.
4. Stained preparation.
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10. 4. METHODS
Study different parts of a compound microscope figure (6) and how to start focusing
the slide under the microscope figure (7).Major parts of compound microscope
are:
a. Eye piece (ocular lens): a magnifying lens with magnification power of
10X.
b. Body tube: Contains mirrors and prisms that transmit the image from the
objective lens to the ocular lens.
c. Objective lenses: Primary lenses that magnify a specimen
• (10X) Low power objective Low power field (L.P.F), used first before
40X and 100X , to focus the slide on the microscope and bring the image to
the ocular lenses.
• (40X)High power objective high power field (H.P.F), used secondly
after the 10X for examination and wet preparation.
• (100X) Oil immersion objective, place a drop of oil is on the slide and
then examine it under the 100X objective lens. It used with stained slides.
d. Stage: Holds the slide in position
e. Condenser: A lens system that condenses light before It passes through the
specimen.
f. Iris diaphragm: controls the amount of light entering the condenser.
g. Coarse and fine adjustment knobs: used for focusing the specimen.
Turning the knob changes the distance between the objective lens and the
specimen.
h. Light: source of illumination, a bulb.
4.2.1. Total magnification power
The image formed by the objective is enlarged by the ocular lens. The total
magnification obtained with any one of the objective lenses is determined by
following:
Total magnification power= Power of the objective lens X Power of ocular lens
e.g., if you are using oil immersion objective (100X), and you know that the power
ocular lens is usually 10X. So, the total magnification power =100 X 10 = 1000.
4.2.2. Resolving power of a microscope
The resolving power of any microscope is a measure of its ability to discriminate
between two adjacent objects. The absolute limit of the resolving power is roughly
the wavelength of the light used to illuminate the specimen. The wave length of
visible light ranges from 400-800 nm.
4.2.3. Field of view
The circular field you see when you look through the ocular lens. The field of view
changes in size at different magnifications.
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11. Figure (6): Component of light microscope
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12. 3. Ad inter
djust rpupillar
distanc
ce.
1. Pllace the specimen
slide on the ob
bject stage
e.
4. Foc
cus 5. ad optiimum
djust
imag contras
ge st
2. A the lamb
Adjust e
brigh
htness.
Figure (7): How to use the microsc
cope at fir (focusi the sp
rst ing pecimen slide).
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13. SAFETY QUIZ :
Q.1. what is the importance of hand washing at the beginning and at the end
of laboratory work?
Q.2. Why we disinfect bench-tops before and after working?
Q.3. Why mouth pipetting is not recommended?
Q.4. Why used syringes and needles discarded in puncture-proof container?
LIGHT MICROSCOPE QUIZ:
Q.1. Explain the use of low power, high power, and oil immersion objectives?
Q.2. What do you understand by total magnification power of
microscope?
Q.3. Which objective is used to focus a specimen?
Q.4. what is the role of condenser and iris diaphragm in focusing specimen?
Q.5. what do you understand by coarse and fine adjustment?
Q.6. Which objective are used to examine wet and stained preparation ?
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14. PRACTICAL No.2
MORPHOLOGY OF BACTERIA
1. OBJECTIVES
1.1. To define the morphological types and arrangements of bacteria
1.2. To differentiate between cocci, bacilli, and spirochaetes.
1.3. To identify different size of bacteria.
1.4. To draw the morphological types and arrangements of bacteria.
2. BACKGROUND
The word morphology means the study of form and structure. Bacteria have a
wide variety of size and shapes. The bacteria that posses cell walls exist in three
distinct basic morphologic forms.
2.1. Basic forms:
2.1.1. Cocci: spherical forms arranged in pairs (diplococcic), in chains of varying
length, and in packet of fours or in groups (clusters). Figure (8)
2.1.2. Bacilli: Rod shaped bacteria may be arranged in pairs , in chains or have
irregular arrangement.
2.1.3. Spiral bacteria (Spirilla): the bacteria that appear snake like having a
series of rigid curves.
2.1.4. Other forms:
2.1.4.1. Spirochaetes: these are SPIRAL bacteria with a series of flexuous curves.
The number of, the depth, and the arrangement of curves vary from one species to
another.
2.1.4.2. Vibrios: curved BACILLI arranged irregularly or In pairs. Figure (8).
2.1.4.3. Filamentous bacteria: long thin bacilli, which may show branching.
2.2. Size
Bacteria are small and measured in terms of microns (1µ = 1/1000 of a
millimeter).Typical bacteria are of 1 µm in diameter, but also vary e.g., anthrax
bacillus 4 to 8 by 1 to 1.5 µm and the whooping cough bacillus 1.5 to 1.8 µm by 0.3
– 0.5 µm.
3. MATERIALS
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15. 1. Microscope.
2. Immersion oil.
3. Lens cleaning paper.
4. 6 Stained slide of: 4.1, 4.2, and 4.3 as follow:
4.1.Cocci:
o Cocci in pairs diplo- cocci e.g. Neisseria gonorrhea Fig 9-1
o Cocci in chains strepto- cocci e.g. Streptococcus pyogenes Fig 9-2
o Cocci in cluster staphylo- cocci e.g. Staphylococcus aureus Fig 9-3
4.2. Bacilli
o Bacilli in pairs.
o Bacilli in irregular arrangement. e.g. Escherichia coli Fig 9-4
o Bacilli in chain. e.g. Bacillus cereus Fig 9-5
4.3. Spirochaetes e.g. Treponema pallidum Fig 9-6
4. METHOD
Examine the stained slides provided and draw illustrative labeled diagram on the
results sheet provided, by consulting Figure (8,9).
Figure (8): Basic bacterial morphology.
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16. Figur
re(9-1): Co in pair = diploc
occi rs coccus, ure(9-2):Co
Figu occi in cha
ains=strepptococcus
s,
Neiss
seria gonor
rrhea .Ligh microsc
ht cope 100X. Strep pyogenes. Light mic
ptococcus p croscope
100X
X.
re(9-3):Coc in clus
Figur cci ster = stap
pylococci, ure(9-4):Ba
Figu acilli in irr
regular ar
rrangemen
n
hylococcus aureus .Light micro
Staph oscope 100
0X. herichia co .Light microscope 100X.
Esch oli m
e(9-5): Bac in cha Bacillu cereus
Figure cilli ain. us ure(9-6): S
Figu Spirochaetes. Trepo
onema
.Light microsco 100X.
t ope lidum .Lig micros
pall ght scope 100XX.
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17. 5. RESULTS
5.1. Cocci:
Slide Morphology Arrangement Drawing Example
In pairs
1 Cocci (diplo-)
In chains
(strepto-)
2 Cocci
In cluster
(stapylo-)
3 Cocci
5.2. Bacilli:
Slide Morphology Arrangement Drawing Example
Random
Bacilli (irregular
4 arrangement)
5 Bacilli Chains
5.3. Bacilli:
Slide Morphology Drawing Example
Spirochaetes
6 (Spiral
bacteria )
Students name:__________________________________________ Student No.______________
UQU Micro. Raniah 1429-1430 17