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PowerPoint Resources
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From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Compound microscope objectives, T. Port

2. Differential Staining
Bacterial
Cell Wall
&
Differential Staining
Image: Bonding structure peptidoglycan, Mouagip; Gram stained slide, T. Port
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Bonding structure peptidoglycan, Mouagip; Other Image Source Unknown

3. Bacterial Cell Wall Function: Shape and protection
Structure: Distinguishes groups of bacteria
Cells that Gram stain
- Gram positive and Gram negative
Cells that resist Gram stain
- Genus Mycobacterium and Norcardia
Stained using Acid-fast staining
techniques
Cells that lack cell walls
Will retain counterstain (second color applied during differential staining).
Images: Gram positive bacteria , Gram-negative bacteria & Acid fast bacteria, all under oil T. Port
From the Virtual Microbiology Classroom on ScienceProfOnline.com

4. Bacterial Cell Wall
___________ is a huge polymer of interlocking chains of alternating monomers.
Provides rigid support while freely permeable to solutes.
Backbone of peptidoglycan molecule composed of two amino sugar derivatives of glucose. The “glycan” part of peptidoglycan:
- N-acetylglucosamine (NAG)
- N-acetlymuramic acid (NAM)
NAG / NAM strands are
connected by interlocking
peptide bridges.
The “peptid” part
of peptidoglycan.
Peptidoglycan
Image: Bonding structure peptidoglycan, Mouagip; Other Image Source Unknown
From the Virtual Microbiology Classroom on ScienceProfOnline.com

5. Bacterial Cell Wall Gram-Positive
- Lipoteichoic acid molecules anchor cell wall to plasma membrane.
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Gram-positive cell wall schematic, Wiki

6. Bacterial Cell Wall Gram-Negative
- Outer membrane is protective, allowing Gram-negative bacteria to survive harsh environments.
- Porins for channels through outer membrane, allow big molecule, such as monosaccharides through.
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Gram-negative cell wall schematic, Jeff Dahl

7. Lipopolysaccharide (LPS)
Q: Why are these differences in cell wall structure so important?
LPS is a lipid-sugar.
Lipid portion is known as ______.
Dead Gram-negative bacteria release lipid-A when this outer membrane disintegrates.
In animals with a Gram-negative bacterial infection, free lipid-A may trigger fever, vasodilation, inflammation, shock and blood clotting.
Killing large numbers of Gram-negative bacteria with antimicrobial drugs releases lots of lipid-A, which can threaten the patient more than the presence of live Gram-negative bacteria.
lipid-A
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Lipopolysaccharide, Wiki

8. Prokaryotes - Cell Wall Gram-Positive & Gram-Negative
Image: Gram-positive cell wall schematic, Wiki; Gram-negative cell wall schematic, Jeff Dahl
From the Virtual Microbiology Classroom on ScienceProfOnline.com

9. Prokaryotes - Cell Wall Gram-Positive & Gram-Negative
Peptidoglycan makes up as much as 90% of the thick, compact cell wall.
Gram-negative
More chemically complex and thinner.
Peptidoglycan only 5 – 20% of the cell wall.
Peptidoglycan not outermost layer, between the plasma membrane and the outer membrane.
Outer membrane is similar to the plasma membrane, but is less permeable and composed of lipopolysaccharides (LPS).
LPS is a harmful substance classified as an endotoxin,
The space between the cell wall and the plasma membrane is called the periplasm.
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Images: Sources Unknown

10. Chemical Warfare Between Species & Selective Toxicity of Antimicrobials
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Penicillium mold growing on plate of Staph, Tom Volk

11. Q: Why are these differences in cell wall structure so important?
1. The bacterial cell wall consists of strands of repeating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) subunits. The NAM subunits have short peptide chains attached to them.
2. The PBP binds the peptide side chains and forms the cross-link with the expulsion of one D-Alanine from one peptide side chain. 3. The PBP dissociates from the wall once the cross-link has been formed.
4. Penicillin is added to the system. It enters the active site of the PBP and reacts with the serine group that is important its enzymatic activity.
5. The beta-lactam ring of penicillin (represented here as the top of the "P") is irreversibly opened during the reaction with the PBP. Penicillin remains covalently linked to the PBP and permanently blocks the active site.
Image: Penicillin inhibition, Wiki; Penicillium mold growing on plate of Staph, Tom Volk
From the Virtual Microbiology Classroom on ScienceProfOnline.com

12. Beta-lactam Antibiotic Resistance
Beta-lactam antibiotics (β-Lactam) are a broad class of antibiotics that all contain a β-lactam ring in their molecular structures. Beta-lactam drugs include penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. These antibiotics work by inhibiting cell wall synthesis in bacteria and are the most widely used group of antibiotics. Some bacteria have developed resistance to β-lactam antibiotics and are able to synthesize an enzyme called β-lactamase, that attacks the β-lactam ring, inactivating the antibiotic.
Penicillin
Cephalosporin
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Images: B-lactam Antibiotics, Action of B-lactamase, Wiki;

13. Meet the Microbes: ________
GRAM-POSITIVE
Facultative anaerobe
coccus-shaped
Coccus-shaped bacteria, which divides in a way that results in grape-like clusters.
Staphylococcus aureus (golden staph), most common cause of staph infections.
Approximately 20–30% of general population “Staph carriers."
S. aureus can cause illnesses ranging from minor skin infections to life-threatening diseases, such as meningitis, Toxic shock syndrome (TSS) & septicemia.
MRSA = Methicillin-resistant Staphylococcus aureus
One of the four most common causes of nosocomial infections, often causing postsurgical wound infections.
S. epidermidis is normal flora which inhabits the skin of healthy humans.
Mannitol Salt
Staphylococcus aureus,
Golden staph
(One of the reasons snot
gets yellow when you are sick.)
Staphylococcus
Our lab friend Stapylococcus epidermidis.
Gram Stain
Image: Mannitol salt plates, T. Port; S. aureus, Janice Haney Carr , PHIL #10046; Gram stain Staph, T. Port
From the Virtual Microbiology Classroom on ScienceProfOnline.com

14. Meet the Microbe: ______ ___
GRAM-NEGATIVE
Facultative anaerobe
bacillus-shaped
Some strains of E. coli inhabit gastrointestinal tracts of warm-blooded animals as normal flora and provide a portion of the microbially-derived vitamin K for their host.
While many strains of E. coli are harmless commensals, of some are human pathogens.
Common cause of bacterial food poisoning and urinary tract infections.
Bacteria must be able to “stick” to cause infection (otherwise, in case of UTI, bacteria would just get peed out).
Bladder lined with proteins, to prevent this. E. coli has fimbriae to help it stick.
MacConkey’s Lactose Fermenter
Escherichia coli
Our lab friend E. coli.
Gram Stain
Images: MacConkey’s, T. Port; E.coli with fimbria, National Library of Science; : E. Gram stain E. coli, T. Port;
From the Virtual Microbiology Classroom on ScienceProfOnline.com

15. Differential Stains Most stains used in microbiology are differential.
Differential stains involve use of more than one dye, so that certain differences between cell type or structures can be distinguished.
Image: Acid fast stain, T. Port
From the Virtual Microbiology Classroom on ScienceProfOnline.com

16. GRAM STAINING PROCEDURE
Crystal violet (1 min) > rinse
Iodine (1 min) > rinse
Acetone Alcohol (10–15 sec) > rinse
Safrinin (1 min) > rinse & blot dry
Distinguishes between two large groups of
microorganisms:
- purple staining, Gram-positive bacteria
- pink staining, Gram-negative bacteria
Q: How does the Gram stain reveal the difference
between Gram+ and Gram- cell wall structure? G + u -
Watch video of
How to Do a
Gram Stain
From the Virtual Microbiology Classroom on ScienceProfOnline.com

17. Gram Stain Examples Staphylococcus epidermidis Escherichia coli
Mixed Sample of S. epidermidis & E. coli
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Images: All Gram stain images by T. Port

18. Bacterial Cell Wall Function: Shape and protection
Structure: Distinguishes groups of bacteria
Cells that Gram stain
- Gram positive and Gram negative
Cells that resist Gram stain
- Genus Mycobacterium and Norcardia
Stained using Acid-fast staining
techniques
Cells that lack cell walls
Will retain counterstain (second color applied during differential staining).
Images: Gram positive bacteria , Gram-negative bacteria & Acid fast bacteria, all under oil T. Port
From the Virtual Microbiology Classroom on ScienceProfOnline.com

19. Mycobacterial Cell Wall
outer lipids
2. mycolic acid
3. polysaccharides
4. peptidoglycan
5. plasma membrane
6 & 7: Molecules involved in evading host immune cells & function.
8. cell wall
Because of waxy cell wall, they can survive exposure to acids, alkalis, detergents, oxidative bursts, lysis by immune system, and many antibiotics.
Image: Mycobacterial cell wall, Ytambe
From the Virtual Microbiology Classroom on ScienceProfOnline.com

20. Meet the Microbes: ________
Mycobacteria colonies Eewwww, looks like ear wax.
GRAM-variable, obligate aerobe, bacillus-shaped
Q: Why Gram variable?
Both __________ and ______________, caused by M. leprae and M. tuberculosis respectively, have plagued mankind for centuries.
Thought that M. tuberculosis and M. leprae evolved from a soil bacterium that infected cows, then made jump to humans about the time of animal domestication, 10,000 years ago.
M. tuberculosis doubles population every hours,
M. leprae doubles population about every 14 days.
Q: What might be the impact of generation
time on the course of the infectious diseases
these microbes cause?
Man with Leprosy
Mycobacterium
A: Mycobacterium produces mycolic acid as part of its cell wall. This is a wax that protects the bacteria.
Tuberculosis
Leprosy
Acid-fast stain
The pink is our lab friend Mycobacterium
smegmatis
Images: TB Culture, Public Health Image Library (PHIL) #4428, Dr. George Kubica; 24 yo man from Norway, suffering from leprosy; Pierre Arents; Acid fast stain of Mycobacteria smegmatis & Staph, T. Port
From the Virtual Microbiology Classroom on ScienceProfOnline.com

21. ACID-FAST STAINING PROCEDURE
Blotting paper
Ziehls carbol fuchsin (3 – 5 min heat) > rinse Acid Alcohol (10 – 15 sec) > rinse
crystal violet (1 min) > rinse & blot dry
For staining cells resistant to Gram staining:
- purple staining, Nonacid-fast cells (NAF)
- bright pink staining, Acid-fast cells (AF)
Q: Specifically what does it reveal about a
bacterium’s cell wall if it is acid-fast?
Create a smear of organism you are testing. Cover smear with a blotting paper.
Saturate paper with Ziehl’s carbol fuchsin (say fyook-sin). Heat 3 – 5 minutes. Remove blotting paper. A
Mycobacteria
Nocardia
Rinse slide with tap water, then decolorize the smear for seconds with acid alcohol. Rinse. + u -
Watch video of
How to Do an
Acid Fast Stain
Apply crystal violet for 1 minute, wash, blot dry.
From the Virtual Microbiology Classroom on ScienceProfOnline.com

22. Acid Fast Stain Examples
Mixed sample of Mycobacterium smegmatis & Micrococcus luteus
Mycobacterium smegmatis
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Images: All acid fast stain images by T. Port

23. Bacterial Cell Wall Function: Shape and protection
Structure: Distinguishes groups of bacteria
Cells that Gram stain
- Gram positive and Gram negative
Cells that resist Gram stain
- Genus Mycobacterium and Norcardia
Stained using Acid-fast staining
techniques
Cells that lack cell walls
Will retain counterstain (second color applied during differential staining).
Images: Gram positive bacteria , Gram-negative bacteria & Acid fast bacteria, all under oil T. Port
From the Virtual Microbiology Classroom on ScienceProfOnline.com

24. Meet the Microbe: _______ _______
Pleiomorphic shaped bacteria with no cell wall.
Cause of primary atypical pneumonia (walking pneumonia).
Transmission: Airborne droplets
Pathogenesis: Bacterial cells attack and destroy ciliated epithelial cells of respiratory track.
Treatment:
Often clears with no intervention.
With no cell wall, these organisms are resistant to the effects of beta-lactam antibiotics.
If antibiotic needed, treatment of choice is Erytrhomycin or Tetracycline (both protein synthesis inhibitors).
Mycoplasma pneumoniae
Diffuse inflammation in both lungs
Dense area of inflammation
Images: Mycoplasma pneumoniae cells, Microbe Wiki; Mycoplasma pneumoniae colonies; X-ray showing atypical pneumonia, PHIL 14372; Typical pneumonia
From the Virtual Microbiology Classroom on ScienceProfOnline.com

25. Confused?
Here are links to resources that further explain bacterial cell wall & differential staining:
Differential Stain Laboratory Main Page on the Virtual Microbiology Classroom of Science Prof Online.
Gram Stain Interactive Tutorial. This is an extremely useful tutorial that shows, step-by-step, what happens in Gram-positive and Gram-negative cells during Gram staining.
Acid-fast Stain Animated Tutorial. The staining procedure depicted in this tutorial differs a bit from how we do it in lab, but this tutorial is still very useful. Shows the steps of the staining procedure and the resulting color of Acid-fast and Nonacid-fast cells.
Videos of differential staining procedures: Gram, Acid-fast, Endospore
Drug Resistant TB: Past, Present & Future, Chang et al (2010) Official Journal of the Asian Pacific Society of Respirology, DOI: /j x
Smart Links
(You must be in PPT slideshow view to click on links.)
From the Virtual Microbiology Classroom on ScienceProfOnline.com

26. Are microbes intimidating you. Do yourself a favor
Are microbes intimidating you? Do yourself a favor. Use the… Virtual Microbiology Classroom (VMC) ! The VMC is full of resources to help you succeed, including:
practice test questions
review questions
study guides and learning objectives
You can access the VMC by going to the Science Prof Online website
Images: Salmonella, Giant Microbes; Prokaryotic cell, Mariana Ruiz