Microscopy: there are a large number of Gram-positive bacilli, but no ...

Lesson 1
Working regime of microbiological laboratory.
The rules of the immersion microscopy.
Methods of the preparation of the smears.
Simple techniques of staining
I. THEORETICAL QUESTIONS 1. The features of design, equipment and working regime of a
bacteriological laboratory.
2. Light microscopy, microscopy with immersion objective, dark- field
microscopy, phase- contrast microscopy, luminescent microscopy, electron
microscopy, scanning microscopy.
3. The rules of work with immersion system of microscope.
4. The main stages of preparation of smears.
5. The dyes used for a staining of bacteria.
6. Simple methods of staining, their practical value.
Principles of health protection and safety rules in the microbiological
laboratory. Design, equipment, and working regimen of a microbiological
laboratory.
A microbiological laboratory usually comprises the following departments:
(1) the preparatory room for preparing laboratory glassware, making
nutrient media and performing other auxiliary works;
(2) washroom;
(3) autoclaving room where nutrient media and laboratory glassware are
sterilized;
(4) room for obtaining material from patients and carriers;
(5) rooms for microscopic and microbiological studies comprising one or two
boxes.
Laboratory rooms should have only one entrance. To facilitate such
procedures as washing and treatment with disinfectants, the walls are
painted with light-colored oil paint or lined with ceramic tiles, whereas
the floors are covered with linoleum.
Equipment of the laboratory. Laboratory furniture should be simple and
convenient. Laboratory tables covered with the special enamel, linoleum, or
other easily disinfecting materials are placed near windows. Safe-
refrigerators are used for storing microorganism cultures.
The bacteriological laboratory must include apparatuses for different types
of microscopy, apparatuses for heating (gas and alcohol burners, electrical
stoves, etc.), incubators, refrigerators, sterilizing apparatuses
(sterilizer, Koch apparatus, Pasteur stove, coagulator, etc.), a
centrifuge, distillator, etc. The used material is rendered safe in the way
which is employed in bacteriological laboratories.
Before starting the work, the premises are disinfected in a way which is
employed for disinfecting the box of microbiological laboratories.
The premises are treated, using disinfectant solutions and bactericidal
lamps.
Preparation and staining of smears, as well as other microbiological
procedures, are performed in a prepared working place. The working table
should contain only those materials and objects which are necessary for the
given examination, namely; the object to be studied (blood, pus, sputum,
faeces, etc.), test tubes or dishes with a culture of microorganisms,
sterile distilled water or isotonic sodium chloride solution, a stand for a
bacteriological loop, a jar with clean glass slides, and felt tip pens.
Other necessary items include a gas or alcohol burner, staining solutions,
a basin with a supporting stand (bridge) for slides, a washer with water,
forceps, filtering paper, a jar with disinfectant solution used for
sterilizing preparations and pipettes.
Rules of the work in the laboratory. 1. The working person at laboratories
should wear medical clothes: coat and cap. Special clothes protect the
worker and also prevent contamination of the studied material with foreign
microflora.
2. Eating and smoking in the laboratory are strictly forbidden.
3. Unnecessary walking about the laboratory, sharp movements, and
irrelevant conversations must be forbidden.
4. The working place should be kept clean and tidy. Bacteriological loops
are sterilized by burning them in the burner's flame; used spatulas, glass
slides, pipettes, and other instruments are placed into jars with
disinfectant solution.
5. After the carrying out of work the nutrient media with inoculated
cultures are placed into an incubator. Devices and apparatuses are set up
in special places. Tables are wiped with disinfectant solution and the
hands are thoroughly washed.
6. If the native material or the culture of microorganisms is accidentally
spilt onto the hands, table, coat, or shoes, they should be immediately
treated with 1 per cent solution of chloramines.
7. Before and after work the surface of the tables are treated with
disinfectant solutions and irradiated with bactericidal lamps. BACTERIOSCOPIC EXAMINATION
The rules of work with immersion system of a microscope.
1. Place the slide specimen-side-up on the stage (the specimen must
be lain over the opening for the light in the middle of the
stage).
2. Put up the condenser using condenser knob.
3. Adjust the total light available by turning the curve mirror and
looking into the ocular. Use the objective with small
magnification (10X).
4. Rotate the nosepiece until immersion objective (black-striped
lens, 90X) takes place above the smear.
5. Drop the immersion oil on the smear.
6. Put down objective into the drop using the coarse focusing knob.
7. Look through the ocular and slowly rotate coarse focusing knob
until an image appears.
8. For clear image use the fine focusing knob.
9. Examine the staining smear and sketch it in the exercises book.
10. Put up the nosepiece and clean the immersion objective with lens
paper or gas wipe.
11. Put down the condenser and the arm of the microscope. Obtaining different magnifications The final magnification is a product of the 2 lenses being used. The
eyepiece or ocular lens magnifies 7X, 10X, 15X. The objective lenses are
mounted on a turret near the stage. They make magnifications: 10X; 40X, and
90X (black-striped oil immersion lens). Final magnifications are as
follows: |Ocular |X |Objective lens |= |Total |
|lens | | | |magnificati|
| | | | |on |
|10X |X |10X |= |100X |
|10X |X |40X |= |400X |
|10X |X |100X (black) |= |900X | Reason for using immersion oil Normally, when light waves travel from one medium into another, they bend.
Therefore, as the light travels from the glass slide to the air, the light
waves bend and are scattered (the "bent pencil" effect when a pencil is
placed in a glass of water). The microscope magnifies this distortion
effect. Also, if high magnification is to be used, more light is needed.
Immersion oil has the same refractive index as glass and, therefore,
provides an optically homogeneous path between the slide and the lens of
the objective. Light waves thus travel from the glass slide, into glass-
like oil, into the glass lens without being scattered or distorting the
image. In other words, the immersion oil "traps" the light and prevents the
distortion effect that is seen as a result of the bending of the light
waves.
II. Students practical activities: 1. Examine the staining smears with immersion system of the microscope.
Sketch the image in the protocol. Resume: Lesson 2
Main researching methods of bacteria morphology.
Preparation of the smears from different cultures of microorganisms.
Simple methods of staining.
I. THEORETICAL QUESTIONS 1. Prokaryote and eukaryote:
a - common properties and differences;
b - features of bacterial cells structure.
2. Chemical composition of prokaryotes:
a - chemical composition of bacteria;
3. Morphology of bacteria:
a - classification of bacteria by the form on cocci, rods, spiral-shaped,
thread-shaped;
b - morphology of cocci and division then in dependence segmentation, to
give examples of pathogenic ones;
c - rod-shaped bacteria (bacteria, bacillus, clostridia) and their locating
in staining, to give examples of pathogenic ones;
d -spiral-shaped forms of bacteria (vibrio, spirilla, spirochaetes) and
give examples of pathogenic representatives.
4. Preparation of the smear from bacterial culture.
5. The simple methods of the staining. 1. PROKARYOTIC CELL STRUCTURE
1. Structure of the envelope:
a. Cell wall (Gram-positive bacteria) or cell envelope (Gram-
negative bacteria)
b. Plasma membrane
c. Capsule or slime layer (may be absent for some bacteria)
2. Cell`s interior:
a. Internal membranous structures (mesosomes)
b. Nucleoid
c. Ribosomes
d. Intracytoplasmic inclusions (may be absent)
3. Outer structures (may be absent):
a. Flagella
b. Pili and fimbriae
Differences between eukaryotic and prokaryotic cells
1. The prokaryotic cell is simpler than the eukaryotic cell at every level,
with one exception: the cell wall may be more complex.
2. The prokaryotic cell is smaller than the eukaryotic cell.
3. The cytoplasm is enclosed within a lipoprotein cell membrane, similar to
the prokaryotic cell membrane.
4. The eucaryotic cell has a membrane-enclosed nucleus. Despite on
eukaryotes the prokaryotes lack a membrane-delimited nucleus. They nave a
nucleoid. The bacterial nucleoid contains the DNA fibrils and is not
separated from the surrounding cytoplasm by membrane.
5. Prokaryotic cells lack autonomous plastids, such as mitochondria, Golgi
apparatus and chloroplasts.
6. Microtubular structures distinguishing for eukaryotic cells are
g