Lab 2 lab 3

1 UQU Micro. Raniah 1429-1430

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.

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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


for all the students and supervisors to follow while they are working in the laboratory(page 2 ).

Clean Hands Save Lives.

Figure ( 1 ): Hand Washing.

Note: Rubbing with soap (step 1 t0 6) should be done for 20 seconds. And repeated if needed tell the hand is clean


2. MEDICAL WASTES

• PAPERS , WATER BOTTLES…etc

A. CONTAMINATED MATERIALS

• GLOVES

• CONTAMINATED PLASTIC LOOPS

• CONTAMINATED WIRE LOOP

• CULTURE PLATES WITH GROWTH

• INFECTED SHARPS (Syringes' Needles, Glass Slides)

BLACK Or TRANSPARENT BAGS

(basket OUTSIDE THE LAB)

Yellow Bags with biohazard sign.

Discard Jars with disinfectant.

Yellow or Red square basket.

Sharp container clearly labeled (NSI).

Red hot (flaming)


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 .

Glass panel

HEPA filter

(decontaminated air)

Figure (2): biological safety cabinets class I


2. The Class II safety cabinets:

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 rubber gloves. Both supply and exhaust air are HEPA-filtered.

HEPA filter

HEPA filter

Glass panel


HEPA filter

Gloves

HEPAfilter

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Figure (3): biological safety cabinets class II

a. These are the best all-round biological safety cabinets for general microbiological usage. Figure (3).

Figure (4): biological safety cabinets class III


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.


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.


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.


Figure (6): Component of light microscope

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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 ?


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


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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5. RESULTS

5.1. Cocci:

Slide Morphology Arrangement Drawing Example

1 Cocci In pairs (diplo-)

2 Cocci

In chains (strepto-)

3 Cocci

In cluster (stapylo-)

5.2. Bacilli:

Slide Morphology Arrangement Drawing Example

4 Bacilli

Random (irregular

arrangement)

5 Bacilli Chains

5.3. Bacilli:

Slide Morphology Drawing Example

6 Spirochaetes

(Spiral bacteria )

Students name:__________________________________________ Student No.______________

Lab 2 lab 3

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