Lecture 3B: Microscopy and Staining. Flashcards
Preparation of Specimens for Light Microscopy
- Smear
- Fixing
- Staining
Smear: Spread a _________________ of material
containing microorganisms over the slide surface. Allow to ____________.
- thin film
- air dry
Spread a thin film of material
containing microorganisms over the slide surface. Allow to air dry.
Smear
Fixing: Process that ________ microorganisms and ____________ them to a microscope slide. Fixing ___________ and ________________ distortion of cells.
- Kills
- attaches
- preserves
- minimizes
Process that kills microorganisms and attaches them to a microscope slide. Fixing preserves and minimizes distortion of cells.
Fixing
Staining: Coloring microorganisms with a dye that emphasizes
certain structures. Before staining a sample, it must be fixed.
Stains are __________ composed of _____________________________________________________________
- salts composed of cation, and anion
The colored ion is called ________
chromphore
two types of dyes
- Basic Dyes
- Acidic Dyes
- Chromophore is in ______________ ions.
Most commonly used dyes. - Bacteria are slightly _______________ charged at pH 7,
- therefore they stain with basic dyes.
- positive
- negatively
Chromophore is in positive ions.
Most commonly used dyes.
Bacteria are slightly negatively charged at pH 7,
therefore they stain with basic dyes.
Basic Dyes
Examples of Basic Dyes and their colors
- Crystal Violet (Purple, purple-blue)
- Methylene Blue (Blue)
- Safranin (Red)
Acidic Dye : Color is in ____________ ions.
Stain the background: ___________________.
Bacteria ______________ stain with acidic dyes.
Used to observe ___________________________________
Minimal distortion because heat fixing is not necessary an dye
is not taken up by cells.
- Negative
- negative staining
- do not
- all shapes, size , and capsules
Examples of acidic dyes and their color
- Eosin (bright pink)
-Nigrosin (Black or dark grey) - India Ink (Black or dark grey)
Types of staining Methods
Simple stains
Differential Stains
Simple staining:
Aqueous or alcohol solution of a ____________________.
Primary purpose is to ________________________________
________________________________.
- single basic dye
- stain entire microorganism to view cell shape and base structures
Procedure for simple staining
- Stain is applied for a certain time and then washed off.
- Slide is dried and examined
- Mordant may be used to increase stain intensity to increase affinity of stain for specimen.
a substance used to set or stabilize stains or dyes
mordant
Differential Stains:
React differently to different types of bacteria.
Can be used to ______________ among different groups of
bacteria
-distinguish
Can be used to Distinguish among different groups of
bacteria
differential Staining.
Two important differential stains used in microbiology
- Gram Stain
- Acidic-Fast Stain
Gram Stain
Developed in 1884 by ____________
Hans Gram, a danish microbiologist
The most useful staining procedure in medical microbiology.
Provides useful information for disease treatment
Gram stain
Gram stain is used to Distinguishes bacteria of two large and medically important
groups:
__________________ bacteria
__________________ bacteria
Gram-positive bacteria
Gram-negative bacteria
why does color change into :
Gram-positive bacteria
Gram-negative bacteria
has something to do on cell wall, and nothing to do with the charge
Steps of Gram Stain
- Primary stain
- Mordant
- Decolorization
- Counterstain
Cover a heat fixed smear with a basic
dye (crystal violet).
- all cells gram-positive and gram-negatuve are stained with crystal violet (appear purple)
Primary stain
After smear is rinsed with water, an iodine mordant solution is applied.
Mordant
Slide is washed with alcohol, which will remove the stain from negative cells but not from gram-positive cells.
Decolorizing
In decolorizing Gram-negative and gram-positive cells will
- Gram-negative will be decolorized, and gram-positive cells will remain purple
Decolorizing meaning (in staining)
- Slide is washed with alcohol which remove the stains from negative cells but not the positive cells.
Alcohol is rinsed off, and safranin is applied, which will stain cell that were decolorized.
- Counterstain
In counterstain Gram-negative and gram-positive cells will
- Gram-negative cells are stained pink/red.
- Gram-positive cells remain, purple
Complete the sentence; Gram-positive cells have very _______ peptidoglycan cell walls, whereas gram-negative cells have very _____ cell walls. Crystal violet easily penetrates both cell types.
- Thick
- Thin
Cells that have very thick peptidoglycan cell walls
- Gram-positive cells
Cells that have very thin cell wall
- Gram-negative cells
Because of its larger size, the crystal violet-iodine complex [CV-I] is not easily removed from gram-_______cells, due to their thick cell wall.
- Gram-positive cells
The CV-I complex is readily washed out of gram-______ cells with alcohol.
- Gram-negative cells
Counterstain (this refers to __________name of stain) only colors gram-______ cells.
- Safranin
- Negative
Where can we apply Gram stain
- Chemotherapy
Gram-positive cells with their very thick peptidoglycan cell walls, are ______________ to penicillins and cephalosporins.
- Susceptible
Gram-negative cells with their thin cell walls and lipopolysaccharide layer are ________________ to these antibiotics.
- Resistant
Not all bacterial cells _____________ with the Gram-stain
- Stain well
Gram-stain only works well on ___________ bacterial cultures, that are actively growing. Therefore, it is best to use cultures that are _________________ hours old.
- Young
- 18-24 hours old
24-28 hours old cultures are _________
- Gram-variable
Those that cannot be group as wither negative or positive.
- Gram-variable
Other name of Acid-fast stain
- Ziehl-Neelsen Stain
Acid-Fast Stain
Modification of a method developed in 1882 by ____________.
- Paul Ehrlich
Used to detect tuberculosis and leprosy causing organisms of the genus _____________ and pathogens of the genus ________________.
- Mycobacterium and Nocardia (Scientific name pagsulat)
These bacteria have ______________ cell walls, which makes them difficult to stain.
- Waxy
Modification of a method developed in 1882 by Paul Ehrlich.
Used to detect tuberculosis and leprosy causing organisms of the genus Mycobaterium and pathogens of the genus Nocardia.
These bacteria have waxy cell walls, which makes them difficult to stain.
- Acidic-fast stain
Steps of Acid-fast Stain
- Primary stain
- Decolorizing
- Counter stain
Cover a heat fixed smear with carbolfuchsin, a red basic dye.
Gently heat for several minutes to increase penetration and retention of dye.
Allow to cool and rinse with water.
- Primary stain
Slide is washed with acid-alcohol.
- Decolorizing
Non acid-fast and acid-fast cells colors in decolorizing:
- Non acid-fast cells will be decolorize.
- Acid-fast will remain red
Acid-alcohol is rinsed off. Methylene blue is applied, which will stain cells that were decolorized.
- Counterstain
Non acid-fast and acid-fast cells colors in counterstain:
- Non acid-fast cells are stained blue, and acid-fast cells remain red
Used to color and isolate specific parts of microorganisms.
- Special stain
Microorganisms that are used to color and isolate specific part in special stain.
- Endospores (endospores stain)
- Capsules (Capsules stain)
- Flagella (Flagella stain)
Most commonly used method is ______________________ endospore stain.
- Schaeffer-Fulton Endospore stain
Endospores are extremely ____________, ____________structures that are formed by some gram-positive bacteria to protect them from ___________________________
- Resistant
- Dormant
- Harsh environmental conditions, heat, drought, chemicals, radiation etc.
Endospore staining:
Ordinary staining methods cannot penetrate the thick __________________.
- Endospore stain
Appearance of cell with endospore
- Pink cell, with green endospore
Steps for Schaeffer-Fulton Endospore Stain
- Primary stain
- Wash
- Counterstain
Used to penetrate endospores
- Malachite green
_________________________ is applied to heat fixed smear and steamed for 5-7 minutes.
- Malachite green
Malachite green is applied to heat fixed smear and steamed for 5-7 minutes.
- Primary Stain
Rinse with water for 30 seconds.
- Wash
_____________ will stain rest of the cell.
- Safranin
Safranin will stain rest of the cell.
- Counterstain
Why is capsules difficult to stain?
- Because they repel most stains, are water soluble and are easily disrupted with harsh environment.
_________________ is used to obtain a dark background (E.g.: India ink or nigrosin).
Cell is stained with a __________________ (e.g.: safranin).
- Negative stain
- Basic Dye
Are gelatinous covers on top of the cell which are important virolence (disease) factors.
Negative stain is used to obtain a dark background (E.g.: India ink or nigrosin).
Cell is stained with a Basic dye (e.g.: safranin).
- Capsule stain
Capsule appearance
- Light halo around stained cell, dark background
Staining procedures are difficult. Usually involves using a mordant and coating the flagellar surface with a dye or metal (eg. Silver)
The number and arrangement of flagella can be used as diagnostic aids.
- Flagella stain
Flagella are appendages used for _______________ that are too thin to be seen easily with a light microscope. Staining procedures are difficult. Usually involves using a _____________ and coating the flagellar surface with a _______ ________________________ The _______________ and _____________ of flagella can be used as diagnostic aids.
- Locomotion
- Mordant
- dye or metal (eg. Silver)
- Number and arrangement.
