Microbiology, Classification of microorganisms, Bacteria, Fungi, Viruses, Isolation methods of microorganisms, Staining techniques, gram staining, Acid fast staining.

1. MICROBIOLOGY
Mrs. S A Wagh
Lecturer,
MVPS’s Institute of Pharmaceutical Sciences,
Adgaon, Nashik.
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2. CONTENTS
• Definition
• Classification of microorganisms
• Shapes and arrangement of Bacteria
• Anatomy of bacterial cell
• Isolation of microorganisms
• Staining techniques
a) Gram staining
b) Acid fast staining
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3. What is microbiology?
It is study of living microorganisms that are microscopic in
size.
micro = tiny
bio = alive/ living
logy = study
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4. Medical Microbiology
• It is study of microscopic organisms that infect man, his reaction
to such infections, the way in which disease is produced and
method for diagnosis, prevention and treatment of such infectious
diseases.
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6. Classification of Microorganisms
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9. BACTERIA
• A bacterium is an unicellular microorganism which does not
contain chlorophyll and does not multiply by true branching.
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11. Shapes of Bacteria
Cocci
Round
or Oval
shaped
bacteria
Bacilli
Rod
shaped
bacteria
Vibrio
Comma
shaped
bacteria
Spirilla
Rigid
and
spiral
shaped
bacteria
Spirochetes
Spiral
shaped
bacteria
look like
Coiled hair
Actinomycetes
Branching
filamentous
bacteria
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12. Arrangement of Bacteria
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13. ANATOMY OF BACTERIAL CELL
• The structure of bacterial cell consists of an outer layer, cell envelope which is differentiated into
an outer rigid cell wall and beneath it is a cytoplasmic membrane which is also called as plasma
memebrane.
• Inside the cell envelope, protoplasm is present.
• Protoplasm is comprised of cytoplasm, cytoplasmic inclusions like ribosomes, mesosomes,
granules, vacuoles and nuclei body.
• In addition to these essential components, some bacteria may contain additional structure.
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15. • Sometimes a bacteria is enclosed into a viscid layer which may be a loose layer or
organised as a capsule. Some bacteria also possess filamentous appendages their
surface. These appendages can be flagella and fimbrae.
• Bacterial nuclei can be seen with microscope after acid or ribonuclease hydrolysis
and subsequent staining. They appear as oval or elongated bodies generally one
per cell.
• Bacterial nucleus does not contain nuclear membrane or nucleolus.
• Bacterial chromosomes are haploid, and replicates by simple fission instead of
mitosis, as in higher animal cells and this is the main basis for classifying bacteria
under the heading of prokaryotes.
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22. ISOLATION OF MICROORGANISMS
• To arrive at a proper diagnosis of various infectious diseases, it is
necessary to know the type and nature of causative microbe.
• For this purpose, specimen of possible infected material are collected.
Mostly, these specimens sent to laboratory are swabs, pus, sputum,
urine, stools, blood, cerebrospinal fluid etc.
• Specimen should be collected before starting the antimicrobial
treatment.
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23. • Materials should be collected:
▫ From the site: most likely to be infected by the suspected microbes.
▫ Stage of the disease: It is also an important factor for collecting
specimen.
▫ Timing: Time of collection of sample is another factor contributing to
successful isolation of causative agent.
• Specimen should be collected in specific quantity in sterilized
containers.
• Urine sample is collected in sterile test tubes or vials.
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24. • Swab from eye, throat, rectum or vagina should be collected by
sterile swab stick that too should be placed in sterilized test tube
immediately after taking sample.
• Sputum should be collected in petridish.
• CSF is collected in sterilized vials. Similarly, blood culture is
collected in bottles.
• After collection, specimens should be delivered to laboratory to
avoid overgrowth of microbes.
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25. METHODS OF ISOLATION OF BACTERIA
• STREAK PLATE
• SPREAD PLATE
• POUR PLATE
• SERIAL DILUTION
• ENRICHMENT CULTURE
• SINGLE CELL ISOLATION BY MICROMANIPULATOR.
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26. STREAK PLATE METHOD
• A plate of solid medium (nutrient agar) is allowed to dry in an incubator for about
30min to dry the surface.
• Then by using bent wire which has been sterilized by heating directly on the flame, is
dipped in an inoculum.
• With this wire the inoculum is streaked across the surface of the agar medium so
that individual cells become separated from each other.
• The inoculum can be streaked on the agar surface by methods as shown in the
diagram.
• These plates are incubated at 370C for about 18-24hrs, after which individual
colonies can be observed on the agar surface.
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28. SPREAD PLATE METHOD
• A drop of diluted sample of culture specimen is placed on the
surface of an agar medium, and this drop is spread over the entire
surface using a sterile bent glass rod.
• These plates are incubated at 370C for about 18-24 hrs, after
which individual colonies can be observed on the agar surface.
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29. SPREAD PLATE METHOD
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30. POUR PLATE METHOD
• In this method, the bacterial sample is mixed with warm agar
medium. The temperature of agar is between 45-500C, to avoid
the damage to microorganisms.
• Sample is poured into petridish and mixed by moving gently as
shown in the diagram, allowed to solidify.
• Plates are then incubated till bacterial colonies grow.
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31. POUR PLATE METHOD
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32. STAINING TECHNIQUES
• After the isolation of causative microbes from infected material,
morphological detail of organism is studied. For morphological
studies, bacteria are stained properly. By staining, bacteria
become clearly visible and can be identified.
• Staining:
Staining is defined as an artificial colouration of a substance to
facilitate examination of tissues, microbes or other cells under
microscope.
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33. SMEAR PREPARATION
• Preparation of a smear is the first step in routine staining procedures.
• A loop full of liquid or fluid specimen or a secretion of bacterial colony
is taken and spread as thin film over required area on a slide.
• Smear is dried in air and then heat fixed by passing through a Bunsen
flame gently.
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36. TYPES OF STAINING
1) SIMPLE STAINING
2)GRAM STAINING
3)ACID FAST STAINING
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37. SIMPLE STAINING
• After fixation of smear, put over any one of stain to be used for ex.
Crystal violet, saffranin, methylene blue, Carbol fuchsin stain.
• Allow stain to react for 30seconds to 3minutes depending on stain
used.
• Wash the smear with gentle stream of cool water.
• Dry between bibulous papers and examine under oil immersion lens of
compound microscope.
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40. GRAM’s STAINING TECHNIQUE
This procedure was described by scientist Christian Gram in 1884.
SIGNIFICANCE:
• By this method, not only shape, size and other structural details are made visible but
this method is also useful to differentiate two major categories of bacteria gram
positive and gram negative.
• To understand how gram staining reaction affects gram positive and gram negative
bacteria based on biochemical and structural differences of their cell wall.
• Also called as “Differential Staining Technique” as it differentiates bacteria into
Gram positive and Gram negative.
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41. Procedure
• Prepare a thin film of smear and dry it.
• Cover the smear with methyl violet stain and wash off residual stain
with excess of grams iodine solution for 1min.
• Smear is then decolorised with spirit.
• Wash the smear quickly with running tap water.
• Cover the smear to dilute carbol fuchsin for 30seconds.
• Wash with tap water and then dry it in air.
• Examine the slide under oil immersion lens.
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42. Observation
1) Gram positive bacteria retain violet colour of methyl violet.
• Ex. Staphylococci, Pneumococci, Bacillus Anthracis, Clostridium tetani,
Cornybacterium diphtheria.
2) Gram negative bacteria are decolourised by spirit, alcohol or acetone
and are stained with a counter stain like carbol fuchsin which gives
them pink stain.
• Ex. E.coli, Salmonella typhi, Meningococci, Heamophilus influenza,
Yersinia Pestis.
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45. Difference between gram positive and
gram negative bacteria
Sr. No. Characteristics Gram positive Gram negative
1. Gram reaction Retain methyl violet stain Decolorised by spirit
and stained by carbol
fuchsin
2. Peptidoglycan Thick layer Thin layer
3. Lipid & lipoprotein content Low High
4. Toxins produced Exotoxins Endotoxins
5. Outer membrane Absent Present
6. Mesosomes More prominent Less prominent
7. Examples Staphylococci, Pnemococci,
Clostridium tetani.
Salmonella typhi, E.
Coli, Meningococci.
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46. ACID FAST STAINING
• This method was discovered by Paul Ehlrich, who observed that
after staining with aniline dyes, tubercle bacilli resist
decolourisation with acids.
• This method was modified two German doctors, Franz Zeihl
(bacteriologist) and Fredrich Neelson (pathologist)
• It is called as Zeihl-Neelson staining technique.
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47. Significance
• The Ziehl-Neelsen stain is a type of differential bacteriological
staining method used to identify acid-fast organisms, mainly
Mycobacterium tuberculosis and M. Leprae.
• It is a modification of acid fast staining and is performed in the
following steps.
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48. Procedure
• Prepare a smear of mucoid part of sputum on a slide and fix it.
• Put strong steaming carbol fuchsin for 5min.
• Wash smear with water.
• Put 20% sulphuric acid for 1min and then wash with water.
• Add methylene blue as counter stain for 30 seconds.
• Wash, dry and observe under microscope.
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49. Observation
• Acid fast bacteria- appear pink or red for ex. Mycobacterium
tuberculosis, Mycobacterium leprae.
• Non acid fast bacteria appears- blue, green.
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