1. Bell Ringer
Identify the two pictures below with Gram reaction, morphology, and arrangement

2. 1) Complete the questions in the lab report and finish Gram staining
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

3. Staining Simple Stains Differential Stains Special Stains Gram stain
Acid-fast stain
Endospore stain
Special Stains
Negative (capsule) stain
Flagellar stain
© 2012 Pearson Education Inc. 3

4. Figure 4.16 Simple stains-overview

5. Use more than one dye so that cells can be distinguished
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. 5

6. 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 under-decolorization
© 2012 Pearson Education Inc. 6

7. 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: Gram-positive cells
remain purple, Gram-negative
cells are pink.
Result: Smear is decolorized;
Gram-positive cells remain
purple, but Gram-negative
cells are now colorless. 7

8. Why do G+ cells appear purple? Why do Gram – cells appear 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?

9. 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. 9

10. Acid-Fast Stain
Why isn’t the Gram stain used to stain Mycobacterium?

11. Staining Differential Stains Endospore stain Bacillus & Clostridium
Stain green
Malachite green and steam
Water
Safranin
© 2012 Pearson Education Inc. 11

12. Staining Special Stains Negative (capsule) Acidic dyes
Repelled by bacteria
Stain environment
Eosin and nigrosin
Capsules
Bacterium
Capsule
Background
stain
© 2012 Pearson Education Inc. 12

13. Cold Calling
How come negative stains do not dye the bacterial cells?

14. Staining Flagellar stain
Mordant binds to flagella and change their color to increase contrast
© 2012 Pearson Education Inc. 14

15. Quiz
15 Points
17 Questions
C. diff and Staining

16. Identification of Microorganisms
Identifying Characteristics
Physical characteristics
Biochemical tests
Serological tests
Phage typing
Analysis of nucleic acids
© 2012 Pearson Education Inc. 16

17. Identification of Microorganisms
Identifying Characteristics
Physical characteristics
Morphology (shape)
Coccus and Bacillus
Physical appearance of bacterial colonies
© 2012 Pearson Education Inc. 17

18. 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. 18

19. Figure 4.23 Two biochemical tests for identifying bacteria-overview
Gas bubble
Inverted tubes to trap gas
Hydrogen
sulfide
produced No
hydrogen
sulfide
Acid with gas
Acid with no gas
Inert 19

20. Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system
Wells 20

21. 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. 21

22. Negative result Positive result Negative result Positive result
Figure An agglutination test, one type of serological test-overview
Negative result
Positive result
Negative result
Positive result 22

23. 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. 23

24. Figure Phage typing
Bacterial lawn
Plaques 24

25. 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. 25

26. 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. 26

27. Figure 4.27 Use of a dichotomous taxonomic key-overview
Gram-positive
cells? No
Yes
Rod-shaped
cells?
Gram-positive
bacteria No
Yes
Cocci and
pleomorphic
bacteria
Can
tolerate
oxygen? No
Yes
Obligate
anaerobes
Ferments
lactose? No
Yes
Can use citric
acid (citrate)
as sole carbon
source?
Non-lactose-
fermenters No
Yes
Produces gas
from glucose?
Produces hydrogen
sulfide gas? No
Yes No
Yes
Produces
acetoin?
Shigella
Escherichia
Salmonella No
Yes
Citrobacter
Enterobacter 27