LESSON № 1

METHODOLOGICAL INSTRUCTION

FOR 2nd COURSE-STUDENTS

of MEDICAL FACULTY

 

Lesson 1 (6 hours)

 

Themes:

I. Working regime of microbiological laboratory. Stains and simple techniques of staining. The main methods of microscopic examination.

ii. Bacteria classification. General characterictics of procaryotic cells. Gram  method.

 

Aim: To familiarize with design, equipment and working regime of a microbiological laboratory. To study the rules of work with immersion system of a microscope. To take possession of smears preparation from pure cultures of microorganisms and pathological material, simple methods of staining and Gram technique.

 

Professional motivation: For making a diagnosis it is necessary to study main rules of smears’ preparation from different tested materials and to examine them under the microscope.

 

Methodology of Practical Class. 9.00-12.00

 

Theme I. Working regime of microbiological laboratory. Stains and simple techniques of staining. The main methods of  microscopic examination.

1. To study the rules of work with immersion system of a microscope.

Positioning the slide

Place the slide specimen-side-up on the stage so that the specimen lies over the opening for the light in the middle of the stage. Secure the slide between (not under) the arms of the mechanical stage. The slide caow be moved from place to place using the 2 control knobs located on the right of the stage.

Adjusting the illumination

Adjust the total light available by turning the flat mirror. Adjust the amount of light coming through the condenser using the iris diaphragm lever located below and to the front of the stage. Light adjustment using the iris diaphragm lever is critical to obtaining proper contrast. For oil immersion microscopy (900X), the iris diaphragm lever should be set almost all the way open (to your left for maximum light).

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:

 

 

Focusing from lower power to higher power:

a. Rotate the 10x objective until it locks into place (total magnification of 100X).

b. Turn the coarse focus control (larger knob) all the way away from you until it stops.

c. Look through the eyepiece and turn the coarse focus control (larger knob) towards you slowly until the specimen comes into view.

d. Get the specimen into sharp focus using the fine focus control (smaller knob) and adjust the light for optimum contrast using the iris diaphragm lever.

e. If higher magnification is desired, simply rotate the 40x objective into place (total magnification of 400x) and the specimen should still be in focus. (Minor adjustments in fine focus and light contrast may be needed.)

f. For maximum magnification (900x or oil immersion), rotate the 40x objective slightly out of position and place a drop of immersion oil on the slide. Now rotate the black-striped 90X oil immersion objective into place. Again, the specimen should remain in focus, although minor adjustments in fine focus and light contrast may be needed.

Cleaning the microscope

Clean the exterior lenses of the eyepiece and objective before and after each lab using lens paper only. (Paper towel or kim-wipes may scratch the lens.) Remove any immersion oil from the oil immersion lens before putting the microscope away.

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.

2. To prepare the smears from agar cultures of Staphylococci or Escherichia coli (the first smear to stain with methylene blue, another one – with fuchsin).

Before making a preparation, glass slides are flamed to ensure their additional degreasing.

In preparing  a smear from bacterial culture grown on a solid medium, a drop of isotonic saline or water is transferred onto the precooled glass. A test tube with the culture is taken by the thumb and the index finger of the left hand. The loop is sterilized in the flame. A cotton-wool plug is pinched by a small finger of the right hand, re­moved from the test tube, and left in this position. The edges of the test tube are flamed and then the loop is introduced into the test tube through the flame. Having cooled the loop against the inner wall of the tube, the loop is touched to the nutrient medium where it meets with the glass wall (if the loop is not sufficiently cooled, it induces cracking and melts the medium). Then the loop is touched to the culture of the microorganisms on the surface of the medium. Then the loop is withdrawn, the edges of the test tube are quickly flamed, the tube is closed with a stopper passed through the flame, and then replaced into the test tube rack. All the above described procedures are made above the flame. The culture sample is placed with the loop into a drop of water on the glass slide and spread uniformly with circular movements on an area of 1– 1.5 cm in diameter, then the loop is flamed.

Then you should do drying an fixation of the smear. 

The fixed preparation is placed, the smear upward, on the support. A dye solution is pipetted onto the entire surface of the smear. With Pfeiffer’s fuchsine the staining lasts 1-2 min, with alkaline solution of Loeffler’s methylene blue or water-alcoholic solution of methylene blue, 3-5 min. Following the staining procedure the dye is dispensed, the  preparation is washed with water, dried between sheets of filter paper, and then examined under the oil-immersion objective.

3. To examine live microorganisms with a help of a dark-field (Leptospirae) and fhase-contrast (Proteus vulgaris) a microscopes.

Theme II. Bacteria classification. General characterictics of procaryotic cells. Gram method.

1. To teach upon microscope and to sketch in an album stains of the main forms of bacteria: spherical (micrococci, diplococci, streptococci, tetracocci, staphylococci, sarcina), rod-shaped (monobacteria, monobacilla, diplobacteria, streptobacteria, Streptobacilla), spiral-shaped (vibriones, spirilla, spirochaetes).

2. To make smears from different cultures and stain them by a simple method.

3. To make smears from an mixture of bacteria (Escherichia coli with Staphylococci) and stain it by Gram method.

 

Break 12.00-12.30

 

Individual Students Program.

You should be  prepared for the practical class using the existing textbooks and lectures. Special attention should be paid to the following questions:

 

Theme I. Working regime of microbiological laboratory. Stains and simple techniques of staining. The main methods of  microscopic examination.

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 smear preparation.

5. The dyes used for a staining of bacteria.

6. Simple methods of staining, their practical value.

 

Theme ii.  Bacteria classification. General characterictics of procaryotic cells. Gram method

1. Prokaryote and eukaryote:

a – common properties and differences;

b – features of bacterial cells structure.

2.   Classification of Prokaryotes (Manual Determinative of Bacteriology Bergey’s):

a – common principles of classification;

b – to make definition of such terms: species, subspecies, serovar, biovar, pathovar, chemovar, strain, clone, population.

3.   Chemical composition of prokaryotes:

a – chemical composition of bacteria;

b – features of chemical composition rickettsiae, spirochaetes, chlamydiae.

4. 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, bacilla, clostridia) and their locating in staining, to give examples of pathogenic ones;

d –spiral-shaped forms of bacteria (vibriones, spirilla, spirochaetes) and give examples of pathogenic representatives.

5. Complex methods of staining, Gram technique:

a – to give definition of complex methods of staining;

b – procedure and mechanism of Gram technique;

c – practical value of Gram staining;

d –  Gram technique by Sinev’s method.

 

 

Seminar discussion of theoretical issues (12.30 – 14.00 hour).

 

Break. 14.00-14.15.

 

Individial student’s work. 14.15-15.00.

Checking practical skills. Checking with quizes  and  constructive questions to verify knowledge level of student who did not do it using “Moodle” system.

 

Test evaluation and situational tasks.

 

Theme I. Working regime of microbiological laboratory. Stains and simple techniques of staining. The main methods of  microscopic examination.

1.     Choose the correct statement:

a –  the scanning microscopy gives the possibility to spectacular three-dimensional image of bacteria; b – luminescence (or fluorescence) is the ability of some objects and dyes to fluoresce upon their exposure to ultraviolet light; c – the dark-field microscopy is useful for detecting unstained bacteria; living objects are visualized in lateral illumination in a dark field of vision; d –  in microscopic examination with the help of immersion objective the latter is immersed in oil (cedar, peachy, “immersiol“, etc.) whose refractive index is close to that of glass; when such a medium is used, a beam of light emerging from the slide is not diffused and the rays arrive at the objective without changing their direction; e –  the wave length of an electron beam is in 10000 times less, than a visible light.

2.     Are these statements correct?

A.   Luminescent  microscopy is the ability of some objects to fluoresce upon their exposure to a beam of electrons; b – ordinary light microscopy allows to achieve the greater magnification then immersion microscopy; c –   electron microscopy provides the flat image of object, and scanning electron microscopy –  three dimensional- image; d –a dark-field  microscopy gives the possibility to examine the staining preparations; e – bacteria in the smear stain in different colors using simple technique of staining; f – Pfeiffer’s fuchsine stains bacteria in red colour, and methylene  blue –  in  blue.

3.     Answers the questions:

A. What is the resolving power of the immersion objective? B.   What is the principle of phase-contrast   microscopy? C. What is the purpose of a condenser at a dark field method? D.  What is the resolving power of an electron microscope? E. Can we increase the resolving power of light microscope by use of ultra– violet beams? F. What beams are used for luminescent microscopy?

4.     Give the answers to questions:

A. Main stages of preparation of a staining smear. B. What mirror are used use for illumination at immersion microscopy?  C. How can we differentiate immersion objective from dry one? D. Why is forbidden to laboratory personnel to bring own things into microbiological laboratory?  E. Why the ring of the bacterial loop should be closed?

 

Theme II. Bacteria classification. General characterictics of procaryotic cells. Gram method

 Chose the correct answers:

1.  Select the correct statements:

a – prokaryote are haploid microorganisms; b- prokaryote contain one gene; c – prokaryote have no mitochondria, chloroplusts and Golgi apparatus; d – prokaryote have no ameba-like motility; e – prokaryote have  nucleoid, haven’t nuclear membrane; f – prokaryote have mesosomes.

4.  To indicate, where are indicated properties of prokaryote and where are eukaryote:

5.  You should get in (paste) percentages of a contents in a bacterial cell:

a – water…; b- dry matter…; c- nucleic acids…; d – polysaccharides…; е – lipids…; f- mineral substances…

4. Select the correct statements: a – the bacteria are subdivided on cocci, rod-shaped, threat-shaped, filiform by their morphology; b – the forms of cocci are bean-shaped, ellipsoidal, lancelet; c – rod-shaped bacteria, which frame spores, are named as the bacilli; d – Spirilla are the threat-shaped form; e – Vibriones have some convolutions; f – the Bacillus and Clostridia is rod-shaped microbes, which frame spores.

 

Student should know:

1.       The principles of health protection and safety rules in the microbiological laboratory.

2.       The rules of  microscopy with an immersion system.

3.       Fundamental principles of a dark-field, luminescent, phase-contrast, electron, scanning microscopes.

4.       .The main forms of bacteria (depends on shape)

5.       The definitions: species, subspecies, serovar, biovar, pathovar, chemovar, strain, clone, population

6.       The mechanism of bacteria Gram staining.

7.       Pathogenic representatives of different types of  microorganisms

 

Student should be able to:

1.       prepare a smears from bacterial cultures;

2.       stain the smears by a simple method (fuchsine, methylen blue);

3.       examine it using an immersion objective of light microscope.

4.       To stain bacteria by Gram technique.

 

The answers to the self– assessments

 

Theme I. Working regime of microbiological laboratory. Stains and simple techniques of staining. The main methods of  microscopic examination.

1. A, b, c, d, e.  2. C, f.  3. A. 0.2 mcm. B. Phase-contrast microscopy is based on the fact that the optical length of the light traveling in any substance depends on its refrac­tive index. Light waves transversing through optically denser sites of the object lag in their phase behind the light waves which do. not have to pass through these sites. The intensity of light in this case remains unaltered but the phase of fluctuation, detected by neither eye nor photoplate, is changed. To increase resolution of the image, the objective is fitted with a special semi-transparent phase plate to create difference in the wave length between the rays of the background and the object. If this difference reaches one – fourth of the wave length, a visually tangible effect occurs when a dark object is clearly seen against a light background (positive contrast) or vice versa (negative contrast) depending on the structure of the phase plate. C. This condenser intercepts the central portion of the parallel beam of rays forming a dark field of vision (illumination in lateral beams); D. It is approximately in 10000 times less than at a light microscopy. E. Yes, we can. F.   Ultraviolet beams. 4. A. Smear preparation, drying, fixation, staining, washing and drying. B. Flat mirror. C. It has black ring (strip), label “MI” (oil immersion), total magnification of 90x, numerous aperture is more then 1. D. For prevention of removal biological  material from microbiological laboratory. E. For keeping a drop of water or  saline solution in it.

 

Theme II. Bacteria classification. General characterictics of procaryotic cells. Gram method

 

 

1. A, b, c, d, e, f. 2. At the left – prokaryote, on the right – eukaryote. 3. Water – 75-85 %, dry matter–25-15 %, proteins – 50-80 % of dry matter, nucleic acid – 10-30 % of dry matter, polysaccharides – 12-18%, Lipids – 10 % of dry matter, mineral substance – 2-14 % of dry mass. 4. A, b, c, d, e, f.

 

Reference:

Basic:

1. Medical microbiology /Patrick R. Murray, 2009, 948 p.

2. Kone man’s color atlas and textbook of diagnostic microbiology /Washington C. Winn. Jr. 2006, 1536 p.

3. Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P.1-10, 35-, 38-45.

http://intranet.tdmu.edu.ua/data/kafedra/theacher/micbio/klymjuk/English/Lectures/Microbiology,%20virology%20and%20immunology/medical/2%20year/Significance%20of%20microbiology%20in%20doctors%20practical%20activity1.ppt

 

Additional:

1. Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P.1-13.

2. E. Jawetz, J.Melnnick, E.Adelberg  Review of Medical Microbiology, 2000,  553 p.

3. W. Levinson, E. Jawetz. Medical Microbiology and Immunology. Examination and Board Review/ Lange medical book. Sixth edition, 2000, 536 p.

4. Bacteriology 330 Lecture Topics: 1997 Kenneth Todar University of Wisconsin Department of Bacteriology, Internet, 2002.

Internet links:

http://www.innvista.com/health/microbes/bacteria/classif.htm

http://www.earthlife.net/prokaryotes/phyla.html

http://web.uct.ac.za/depts/mmi/lectures/bactax/ppframe.html

http://www.gsbs.utmb.edu/microbook/ch003.htm

http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/dental/tutorials/classification/introduction.html

http://www.microbiol.org/WPaper.Gram.htm

 

The methodical instruction has been done:

by prof. Sergey I. Klymnyuk, ass. prof. Olena V. Pokryshko

 

Methodical instruction was discussed at the Department sitting

24.09.2013. Minute № 3