1. Gram negative vs. Gram Positive
Physical differences & clinical signifigance.

2. Gram- positive bacteria
Figure 4.8b

3. Gram-negative bacteria
Figure 4.8a

5. Gram-Positive cell walls
Teichoic acids:
Lipoteichoic acid links to plasma membrane
Wall teichoic acid links to peptidoglycan
May regulate movement of cations
Polysaccharides provide antigenic variation
Antigenic variation – when a bacteria (or protist) can change its surface proteins to evade a host’s immune response.

6. Gram Negative: Outer Membrane (OM)
A bilayer membrane found in gram-negative bacteria
Forms the outermost layer of the cell wall; is attached to the peptidoglycan by a continuous layer of lipoprotein molecules
Proteins called porins form channels through the OM (transports some proteins)
OM has surface antigens and receptors
Does prevent some molecules from entering (e.g. some antibiotics), but generally isn’t that selective

7. Gram-Negative Outer Membrane
Figure 4.13c

9. Damage to Cell Walls Lysozyme digests disaccharide in peptidoglycan.
Penicillin inhibits peptide bridges in peptidoglycan. (effective on Gram-positive cells) .

10. Bacterial Membranes

11. Gram-Positive Cell Wall
Gram-Negative Cell Wall
Thicker layers of peptidoglycan
2-ring basal body
Teichoic acid
Disrupted by lysozyme
Penicillin sensitive
More penetrable
Thinner layer of peptidoglycan
4-ring basal body
No teichoic acid; porin proteins
Endotoxin
Tetracycline sensitive
Less penetrable
Figure 4.13b–c

12. Exotoxins vs. Endotoxins
EXOTOXIN ANIMATION
ENDOTOXIN ANIMATION

13. Types of staining techniques:
Simple Staining:
use of a single stain
Differential staining:
Use of 2 contrasting stains
Separation into groups:
gram stain
acid stain
Visualization of various structures: flagella, capsule, spore or nuclear stain
Gram stain – developed in 1884 by Hans Christian Gram.
Clinically important because it gives us a way to classify bacteria by their cell wall (knowing about the cell wall make-up can help us to treat a disease with the appropriate drug.)

14. The Gram Stain
Gram-Positive
(b) Gram-Negative
Table 4.1

15. Techniques of Light Microscopy
Wet mounts: A drop of medium containing organisms is placed on slide and used to view living microorganisms
Smears: Microorganisms are spread onto the surface of a glass slide and used to view destroyed organisms
Heat fixation: destroys the organisms, causes organism to adhere to slide, and alters organism to accept stains (dyes)

16. animation Outline of Gram Stain procedure
Step 1: applying primary stain
Step 2: apply mordant
animation
Step 3: apply decolorizing agent
Step 4: apply counterstain

17. Gram Positive and Gram Negative Cells

18. Gram Stain Color of Gram-positive cells Gram-negative cells
Primary stain:
Crystal violet
Purple
Mordant:
Iodine
Decolorizing agent:
Alcohol-acetone
Colorless
Counterstain:
Safranin
Red

19. Micrograph of Gram-Stained Bacteria
Figure 3.12b

20. The Gram Stain Mechanism
Crystal violet-iodine crystals form in cell
Gram-positive
Alcohol dehydrates peptidoglycan
CV-I crystals do not leave
Gram-negative
Alcohol dissolves outer membrane and leaves holes in peptidoglycan
CV-I washes out

21. Distinguishing Bacteria by Cell Walls
Gram-positive Bacteria have a relatively thick layer of peptidoglycan (60-90%)
Gram-negative Bacteria have a more complex cell wall with a thin layer of peptidoglycan (10-20%)
Acid-fast Bacteria is thick, like that of gram-positive bacteria, but has much less peptidoglycan and about 60% lipid (mycolic acid)… Mycobacteria (TB and leprosy)

22. Acid-Fast Bacteria
Found in bacteria that belong to the genus, Mycobacterium sp. (e.g. tuberculosis, leprosy)
Cell wall is mainly composed of lipid, makes them very hardy. Also has slow diffusion = slow growth rate for many (eg: 20 days for M. leprae)
Lipid component is mycolic acid
Acid-fast bacteria stain gram-positive

23. Acid-Fast Stain Color of Acid-fast Non–Acid-fast Primary stain:
Carbolfuchsin
Red
Decolorizing agent:
Acid-alcohol
Colorless
Counterstain:
Methylene blue
Blue

24. Acid-Fast Bacteria
Figure 3.13

25. Q&A
Acid-fast staining of a patient’s sputum is a rapid, reliable, and inexpensive method to diagnose tuberculosis. What color would bacterial cells appear if the patient has tuberculosis?

26. Why doesn’t a negative stain color a cell? 3-7
Why is fixing necessary for most staining procedures? 3-8
Why is the Gram stain so useful? 3-9

27. Normal Microbiota
Bacteria were once classified as plants, giving rise to use of the term flora for microbes
This term has been replaced by microbiota
Microbes normally present in and on the human body are called normal microbiota

28. Normal Microbiota on Human Tongue
Figure 1.7

29. Normal Microbiota Normal microbiota prevent growth of pathogens
Normal microbiota produce growth factors such as folic acid and vitamin K
Resistance is the ability of the body to ward off disease
Resistance factors include skin, stomach acid, and antimicrobial chemicals

30. Biofilms Microbes attach to solid surfaces and grow into masses
They will grow on rocks, pipes, teeth, and medical implants

31. Biofilms
Figure 1.8