Practical class

Theme: MORPHOLOGY AND STRUCTURE OF BACTERIA. STAINING OF

BACTERIA METHOD BY GRAM.
The relevance of theme. Study of surface structures of bacteria - the important stage
ofstudies after the theme «Morphology and physiology of microorganisms». A
capsule, cell wall, cytoplasmic membrane - are the organoids of bacterial cell, which
determine its properties, also pathogenic. A capsule has an antigen characteristic,
protects microorganismfromunspecific factors of protection of microorganism and
from unfavorable conditions of environment. A cell wall presents by itself
biogeteropolimer of difficult chemical structure, which covers all surface of cell.
Composition of thisbiogeteropolimer is not identical for different bacteria. Depending
on the structure of cell wall bacteria are divided into two enormous groups: gram-
negative (gr -) and gram-positive( gr +), which differentiate on staining of smears
by the method of Grame. LPS of cell wall has antigen and toxic characteristics.
Peptidoglyca of cell wall is a «target»for the action of penicillin and lisocim.
Formation ofL-forms is a reason of resistance to the antibiotics. L –form of different
bacteria plays an important role in pathogeny of many infectious diseases.
Cytoplasmic membrane makesvitally –important functions, thedisturbance of which
brings a bacterial cell to death. The study of surface structures of bacterial cells
allows doctor to have imagination about antigen, pathogenic and physiological
properties of exciters of infectious diseases. Especially actuality is a questionof
forming the mechanism of resistance bacteria to antibiotics. The meaning of this
questions allow to doctor make the correct chose of treatment.
Purpose(general): to know how to estimate and analyze the results of identification
the agent of infectious diseases in depends on condition of surface structures.
Concrete aims – to know how:
1.To prepare the fixed preparation from bacterial structures.
2.To differentiate at the microscopy Gr+ and Gr- microorganisms.
3.To stain a smear by the method of Gram.
4.To explain the role of surface structures of bacteria in diagnostic and pathogeny of
infectious diseases.
Base level of knowledge - abilities:
1.To know how to explain the structure of eukaryotic cell and function of cell’s
organelles (department of biology).
2.To interpret the biological role of chemical matters which are included in
composition of cell structures (departmentof biology).
3.Be able to work with a microscope, to know an immersion microscopy (department
of physic and microbiology).
4.To know how to schematically represent the structural elements of bacterial cell.
5. To explain the differences of prokaryotic and eukaryotic cells.
In order that you could realize does the initial level of your knowledge - abilities
match to necessary requirements we suggest to make the row of tasks:
Task1. Name the motional organelles of eukaryotic cells.
Task2.Classification of chemical matters, which structures of cells of eukaryotic and
prokaryotic are composed.
Task 3. Name basic parts a microscope. Explain the direction of rays in optical part of
microscope. What name do the details of optical part of microscope have?
Task4. Do the preparations of bacteria need the fixation, if they are studied with help
of dark-field microscopy?
Task5. In what cases do the chemical methods of fixation used?
Information for addition to knowledge-abilities it is possible to find in the followings
textbooks:
1. Berezov T.T. Korovkin V.F. Biologycalchemictry. M.: Medicine.- 1998.
2. Biology (under red.V.N.Yarygin.G.High School. - 2004.-t.1. P- 431).
3. A.A.Vorobyov and coauthors. Microbiology. : M,1999, P.3-19.
4. Medical microbiology, virology and immunology / Under red.B.Borisov.
A.N.Smirnova. M.: Medicine, 1994.
5. Rukovodstvotopractic works mikrobiologii. /Under red. L.B.Borisov. M.:
Medicine, 1984.
Theoretical questions, on base of which it is possible to make a special purpose types
of activity:
1.Capsule, structure and functions.
2.A cell wall - structure and functions.
3.Cytoplazmatic membrane - structure and functions.
4.Structure and meaning of flagellums.
5.How does the bacteria are classified depending on an amount and location of
flagellums.
6.What does the phenome of plazmoliz consist in? Practical use.
7.Principle of method byGrame staining.
8.Differences of Gr+ and Gr- of bacteria
Literature for learning of knowledges-abilities after this theme:
1. Medial microbiology, virology and immunology / Under red. L.B.Borisov,
A.M.Smirnova. M: Medicine,2005. p.30-32.
2. Guidance to practicum for microbiology. / Under red. L.B.Borisov. M.:
Medicine, 1984. P.96-104.
3.A.A.Vorob'ev and coauthors. Microbiology. -M. Medicine.1998. p.3-19.
4. Shlegel G. General microbiology M.:World. 1972.P-476.
5.Medical microbiology, virology and immunology / Under red.Vorobyov.A.A.-
M.:OOO”Medical informative agency”, 2008-P.36-42.
6.Practicum of microbiology. (under red. A.I. Netrusova. M.: Publishmentcentr
“Academy”, 2005-P.74-75.
This content must provide reaching goals of learning, which the structure of theme is
facilitated: Surface structure of bacteria cell. Staining by Gram method (addition 1).
Materials and equipment: cultures of microorganisms, microscope slide, sterile
saline solution, staining set for Gram method (gentian violet,Lugol’s solution, spirit,
diluted fuchsine, filter paper), microscope, bacteriological loops, sterile tubes with
slant agar.
Indicative basis of action at performing the practical work.
1.Prepare fixed smears with pathological material or with bacteria mix. Using
practicum (N2 from the literature list), chose from the suggested set of equipment
instruments and reactive, which is need for staining. Add to protocol the Gram
method of staining.
2.Stain prepared smears by Gram, microscopy with immersion, pay attention on
bacteria staining, identify the morphological group. Add to the protocol pictures, sign
the Gr+ and Gr- microorganisms.
3.Whatch under the microscope the demonstrative preparations of different
microorganisms. Identify it’s method of staining by Gram and morphological group.
Add to the protocol pictures with signatures.
4.Compere the characteristics of Gr+ and Gr- bacteria and complete the table in the
protocol.
After learning the theoretical questions and motion of conducting of experiment
it is offered to you to make teaching task.
1.From a peritoneal liquid was prepared and painted smear. At the microscopy of
smear there areviolet cocci which are located wrong accumulations, and sticks of
middle sizeswith the rounded ends, located chaotically and stained into a rose color.
What can you say about the microflora of peritoneal exudate? What method of
staining was used in this case and what main stages does it has?
2.For staining the phlegm of sick with suspicion on pneumonia the following staining
was used: solution of gentian violet, Lugol’ssolution,96% spirits, water fuchsine.
What mean of staining was used in this case?
 Addition1
Graphological structure of theme: Morphology and structure of bacteria. Staining by
Gram method.

Surfacestructuresofbacteriacell

Capsule Cell wall Cytoplasmic Flagellum Fimbriae

membrane

monotrikh,
Functions lipoprotein peritrikh,
amfitrikh,
enzymes lofotrikh,
rigidity
flagellin
Pathogenic, antigenic properties
Polysacharides, polypeptides

Gr+ bacteria

Gr- bacteria

styrenes
elastance
functions Fineproteinaceou
Protective function

sends
protective
Biosyntesis of
CW adhesion
Transport
antigenic
conjugation
Replication of
Microcapsule
adhesive DNA

spore-
Peptidoglican, Single- formation
teichoicacids, a protein layerpeptodoglican,
LPS-
- a ribonucleateof a antigenicandtoxiferou
magnesium, RNA: DNA sproperties.
= 8:1. Coloring ColoringbyGraminred
byGram: inXing –

Consistsofmicrofibrils Protoplast, spheroplast,

L-form
mucopolysaccharides
Resistancetoantibiotics
Protective function
 Addition 2
Example of protocol to practical class.
Theme: MORPHOLOGY AD STRUCTURE OF BACTERIA. STAINING OF
BACTERIA METHOD BY GRAM.
Task1. Stages of preparing the preparations by Gram method.
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Task2. Staining the mix of bacteria b Gram.

Picture 1 Picture 2 Picture 3

Task3. Gr+ and Gr- microorganisms

Picture 4 Picture 5 Picture 6

Task4. What difference between Gr+ and Gr- bacteria?
Gr+ Gr-

Date__________ Signature of teacher_______________