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Methods of Microbiology: Staining Media Micros

This document discusses various microbiological techniques including staining, culture media, and microscopy. Staining methods are used to increase contrast and identify cell structures. Different types of media such as complex, selective, and differential media are used for culturing microorganisms. Microscopy techniques including light microscopy, fluorescent microscopy, and electron microscopy are used to visualize microbes with different magnification capabilities and resolutions.

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32 views

Methods of Microbiology: Staining Media Micros

This document discusses various microbiological techniques including staining, culture media, and microscopy. Staining methods are used to increase contrast and identify cell structures. Different types of media such as complex, selective, and differential media are used for culturing microorganisms. Microscopy techniques including light microscopy, fluorescent microscopy, and electron microscopy are used to visualize microbes with different magnification capabilities and resolutions.

Uploaded by

dean_tsipi
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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Methods of Microbiology

Staining
Media
Microscopy
Staining
• Increase contrast of microorganisms
• Classified into types of stains
– Simple stain:

– Differential stain:

– Structural or special stains


Dyes
• Organic salts with positive and negative
charges
• One ion is colored -chromophore
• Basic dye: positive ion is colored
– MeBlue+ Cl-
• Acidic dye: negative ion is chromphore
Basic Dye
• Works best in neutral or alkaline pH
• Bacterial cell wall has slight negative
charge at pH 7
• Basic dye (positive) attracted to cell wall
( negative)
• Crystal violet, methylene blue, safranin
Acidic Dye
• Chromophore repelled by negative cell
wall
• Background is stained, bacteria are
colorless
• Negative stain-look at size, shape
• Acidic dye will stain bacteria if grown at
lower pH
• Eosin, India ink
Simple Stains
• One dye, one step

• Direct stain using basic dye

• Negative stain using acidic dye


Differential Stains
• More than one dye
• Gram stain, acid fast

• Primary dye
• Decolorizing step

• Counter stain
Special/ Structural Stains
• Identify structures within or on cells

• Different parts of cell are stained different


colors
Media
• Culture media
• Inoculum

• Culture

• Pure culture

• Million cells to be visible


Living vs Nonliving
• Viruses, few bacteria
• Living host-eggs, tissue cells
• Mycobacterium leprae –armadillos
• Most microbes grow on nonliving media
Synthetic or Chemically Defined
• Exact chemical composition known
• Chemoheterotrophs
– Glucose-carbon source & energy source
– Supplies chemical requirements
Complex Media
• Used for most chemoheterotrophs
• Bacteria and fungi
• Cannot write formula for each ingredient
• C,N,energy, S requirements
• Vitamins, other growth factors
Complex Media
• Nutrient broth –liquid form

• Nutrient agar –solid form


– Plate

– Deep

– Slant
Anaerobic Methods
• Reducing media

• Anaerobic jar

• Use both in lab


Candle Jar
• Reduce oxygen levels
• Provides more CO2
• Microaerophilics
Selective and Differential Media
• Selective

• Differential
Filtration
• Passage of liquid through screen device
• Pores small enough to retain microbes
• Sterilize heat sensitive materials

• Negative
• Positive
• HEPA hoods & TB rooms
Autoclave
• Uses temperature above boiling water
• Steam under pressure
• Preferred method unless material is
damaged
• Higher the pressure, higher the
temperature
• Need direct contact with steam
• 15 psi at 121 C for 15 mins
Autoclave
• Prions- protein only

• Flash sterilization-at 134 C for 3min

• Packaging
• Use of indicators
Compound Microscope

• Assigned scope
• Know parts & functions
• Proper use & care of scope
Microscope
• Use of light ( visible or UV) or electrons

• Lenses to magnify

• Total magnification of compound scope


Lenses
• Parfocal

• Working distance
Resolution
• Ability to distinguish 2 objects as separate
and distinct

• Dictated by physical properties of light

• Limit is 0.2um for our light scope


Light Scope
• Simple vs compound
• Source of illumination

• Visible light has average wavelength of


550nm or 0.55 um
– Enters condenser lenses
– Focused into a cone
Light Path
• Passes through opening in stage to slide

• Light enters objective lens


– Image magnified by ocular lens
Contrast
• Improves image detail
• Difference in light intensity

• Bacteria are colorless


• Need to increase artificially by staining

• Contrast is property of specimen


Resolution
• Distinguish detail within image
• Property of lens system, measured as
resolving power
• Closest that 2 points can be together and
still seen as separate
• RP = wavelength of light
2 X NA
Resolving Power
• Function of numerical aperture: NA

• Function of wavelength of light

• Refractive index of material between


objective lens & specimen
Oil Immersion Lens
• Light bends (refracts) as it passes from
glass into air

• Use oil between slide and 100x lens

• Increases resolution
Oil Immersion Lens
• Lens captures more light

• Shortest working distance

• Summary: increased resolution


Fluorescent Microscope
• Used to view antigen antibody reactions

• Specimen tagged with fluorescent dye

• Ocular lens fitted with filter that permits


longer wavelengths & blocks shorter ones

• UV light(230-350nm)
Electron Microscopy
• Uses electrons as source of illumination

• Wavelength of electrons is dependent


upon voltage of electron beam

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