Download Staining

Bell Ringer
• Identify the two pictures below with Gram
reaction, morphology, and arrangement
In The Next 30 Minutes…
• 1) Complete the questions in the lab report and
finish Gram staining
– Collect
• 2) Work on handout
– P. 68 in textbook
– Describe acid-fast, endospore, flagella, and
negative staining procedures
– Homework if not complete
• You will have a 15 point quiz at the end of class
covering the material taught today
Staining
• Simple Stains
• Differential Stains
– Gram stain
– Acid-fast stain
– Endospore stain
• Special Stains
– Negative (capsule) stain
– Flagellar stain
© 2012 Pearson Education Inc.
Figure 4.16 Simple stains-overview
Staining
• Differential Stains
• Use more than one dye so that cells can be
distinguished
– Gram stain
– Acid-fast stain
– Endospore stain
© 2012 Pearson Education Inc.
Staining
• Differential Stains
– Gram stain
– Gram (+) & Gram (-)
–
–
–
–
Primary stain- crystal violet
Mordant- iodine
Decolorizing agent- acid alcohol
Counterstain- safranin
– Young cells
– Thin smear
– Avoid over over-decolorization and underdecolorization
© 2012 Pearson Education Inc.
Figure 4.17 The Gram staining procedure-overview
Slide is flooded with crystal
violet for 1 min, then rinsed
with water.
Slide is flooded with iodine
for 1 min, then rinsed with
water.
Result: All cells are stained
purple.
Result: Iodine acts as a
mordant; all cells remain
purple.
Slide is flooded with solution
of ethanol and acetone for
10–30 sec, then rinsed with
water.
Slide is flooded with safranin
for 1 min, then rinsed with
water and blotted dry.
Result: Smear is decolorized;
Gram-positive cells remain
purple, but Gram-negative
cells are now colorless.
Result: Gram-positive cells
remain purple, Gram-negative
cells are pink.
Cold Calling
• Why do G+ cells appear purple?
• Why do Gram – cells appear pink?
• What would result if you did not decolorize during
the gram stain?
– How would the cells appear?
• Why would a G+ cell appear purple and pink after
Gram staining?
Staining
• Differential Stains
– Acid-fast stain
– Mycobacterium & Nocardia
– M. tuberculosis and M. leprae
– Do not gram stain due to waxy cell walls
– Appear red or pink
– Carbolfuchsin
– Acid-alcohol
– Methylene Blue
© 2012 Pearson Education Inc.
Acid-Fast Stain
• Why isn’t the Gram stain used to stain
Mycobacterium?
Staining
• Differential Stains
– Endospore stain
– Bacillus & Clostridium
– Stain green
– Malachite green and steam
– Water
– Safranin
© 2012 Pearson Education Inc.
Staining
• Special Stains
– Negative (capsule)
– Acidic dyes
–
–
–
–
Repelled by bacteria
Stain environment
Eosin and nigrosin
Capsules
© 2012 Pearson Education Inc.
Bacterium
Capsule
Background
stain
Cold Calling
• How come negative stains do not dye the
bacterial cells?
Staining
– Flagellar stain
– Mordant binds to flagella and change their color
to increase contrast
© 2012 Pearson Education Inc.
Quiz
• 15 Points
• 17 Questions
• C. diff and Staining
Identification of Microorganisms
• Identifying Characteristics
–
–
–
–
–
Physical characteristics
Biochemical tests
Serological tests
Phage typing
Analysis of nucleic acids
© 2012 Pearson Education Inc.
Identification of Microorganisms
• Identifying Characteristics
– Physical characteristics
– Morphology (shape)
– Coccus and Bacillus
– Physical appearance of bacterial colonies
© 2012 Pearson Education Inc.
Identification of Microorganisms
• Identifying Characteristics
– Biochemical tests
– Utilize or produce certain chemicals
– Ferment various carbohydrates
– Produce hydrogen sulfide gas as a waste product
– Used in labs to identify pathogens
– Color change indicates metabolic reaction
– The pattern of colors correlates to a specific pathogen
© 2012 Pearson Education Inc.
Figure 4.23 Two biochemical tests for identifying bacteria-overview
Gas bubble
Acid with gas
Inverted tubes to trap gas
Acid with no gas
Inert
Hydrogen
sulfide
produced
No
hydrogen
sulfide
Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system
Wells
Identification of Microorganisms
• Identifying Characteristics
– Serological tests
– The study of serum (liquid portion of blood)
– Antigen antibody reactions
– Antibodies- part of immune system that bind
specifically to a antigen
– Agglutination test- antiserum mixed with target
antigens
– Clumping occurs if antigens as present
© 2012 Pearson Education Inc.
Figure 4.25 An agglutination test, one type of serological test-overview
Negative result
Positive result
Negative result
Positive result
Identification of Microorganisms
• Identifying Characteristics
– Phage typing
– Bacteriophages (phages)- viruses that infect bacteria
– Phages are specific to their host
– One bacterial strain is susceptible while another
is not
– Bacteria is lawned on media
– Drops of different phage solutions are placed on
plate
– Plaque- clear zones on bacterial lawn
© 2012 Pearson Education Inc.
Figure 4.26 Phage typing
Bacterial lawn
Plaques
Identification of Microorganisms
• Identifying Characteristics
– Analysis of nucleic acids
– Sequencing of nucleic acids
– Polymerase chain reaction (PCR)
– Examine G + C ratios
– 20%-80% in prokaryotes
© 2012 Pearson Education Inc.
Classification and Identification of Microorganisms
• Taxonomic Keys
– Dichotomous keys
– Series of paired statements where only one of two
“either/or” choices applies to any particular
organism
– Key directs user to another pair of statements, or
provides name of organism
© 2012 Pearson Education Inc.
Figure 4.27 Use of a dichotomous taxonomic key-overview
Gram-positive
cells?
No
Yes
Gram-positive
bacteria
Rod-shaped
cells?
No
Yes
Can
tolerate
oxygen?
Cocci and
pleomorphic
bacteria
No
Yes
Ferments
lactose?
Obligate
anaerobes
No
Yes
Non-lactosefermenters
Can use citric
acid (citrate)
as sole carbon
source?
No
Yes
Produces gas
from glucose?
No
Shigella
Produces hydrogen
sulfide gas?
No
Yes
Escherichia
Yes
Produces
acetoin?
No
Citrobacter
Salmonella
Yes
Enterobacter