Bacteriology (trans 4) Flashcards
(102 cards)
1
Q
HISTORY OF BACTERIOLOGY
1665: Robert Hooke: first observation of cells
1673: Antonie Van Leeuwenhoek: “Father of Microbiology,” first observed single-celled live organisms
1735: Carolus Linnaeus: “Father of Taxonomy”, invented the modern biological naming scheme of binomial nomenclature
1798: Edward Jenner: invented the small pox vaccine
1857-64: Louis Pasteur: fermentation, disproved spontaneous generation, developed the Biogenesis Theory: life forms came from other life forms; Pasteurization
1867: Joseph Lister: came up with Aseptic surgery following the work of Louis Pasteur
1876: Robert Koch: Germ theory of disease building on Louis Pasteur’s work
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1884: Elie Metchnikoff: best known for his discovery of the contribution of phagocytosis of the macrophages in the immune system
1885: Hans-Christian Gram: developed the Gram-staining method. (Although the gold standard is culture, gram-staining is used because we want to give empiric treatment. Sometimes we have enough data to diagnose something, and the gram-staining of the specimen matched the probable diagnosis. We can give treatment immediately because culture takes at least 3 days. If not given immediate treatment, the patient can have ascending infection.)
2
Q
INOCULATION, INTUBATION, AND ISOLATION
Some tools in Microbiology:
1. Petri Dish/Plates: where cultures are grown
2. Autoclave: This is operated under the principle of moist, heat, temperature, and pressure. For example, having 121oC, 18 psi (pounds per square inch), for 15-20 minutes, with moisture under control.
3. Inoculating loop and needles: for streaking, transferring of specimen, etc
A
Steps in culturing organisms:
1. Inoculation
o Inoculate the culture in the proper medium
2. Incubation
o Incubate at 37oC for 18-24 hours
**Why 37oC? Most of the pathogenic bacteria that we will know later are mesophilic, meaning they love 37oC, and that means exactly our body temperature.
**What does the immune system do in cases of infection? The core temperature of the body will be raised to prevent bacteria from multiplication. Fever is both good (helps us combat infection) and bad.
3. Isolation
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Q
INOCULATION, INTUBATION, AND ISOLATION - Steps in Inoculating
In inoculating, do not streak too soft (the organism may not grow) or too hard (the agar may be destroyed)
Primary streaking seeks to:
o Isolate the colonies
o Know if what we are dealing with is a pure culture or a mixed culture & identify if the infection is polymicrobial
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A streaking pattern in streak-plate technique. After doing the first streak, the plate is rotated approximately 45o then streak again with another culture, and so on. Sometimes the streaks are used to semi-quantitate (light, moderate, heavy growth), but usage of those terms may indicate sepsis, so we use positive or negative growth.
4
Q
INOCULATION, INTUBATION, AND ISOLATION - Isolation
Done depending on what was found out in inoculation.
o Contaminated culture: characterized by the presence of culture not in the lines of streaking
o Colonies are like blebs or bump in the media
Followed by getting a subculture and identifying the microbe
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a sample unknown testing (for Staphylococcus aureus):
- A boil, carbuncle, or caruncle (abscess) in the right antecubital fossa
- We do gram stain and we saw round bacteria (cocci), and the last reagent shows violet color (gram positive cocci in clusters)
- Catalase testing: A positive result is effervescence or bubble formation after adding H2O2.(Staph is catalase +)
- Coagulase testing: isolated colony and plasma are placed together in a test tube. A positive result is clot formation. (if there is no clot, it may be Staph epidermidis or Staph saprophiticus; a clot formation means Staph aureus)
- Antibiotic testing: Staph saprophiticus is resistant to novobiocin while Staph epidermidis is sensitive to novobiocin.
- Culture in a selective medium: Mannitol salt agar since Staph aureus would like to grow at a high salt concentration. It ferments mannitol that’s why the peach color becomes yellow.
* Each organism will have its own algorithm. In our unknown testing, we will do something like this.
5
Q
INOCULATION, INTUBATION, AND ISOLATION - Effective antimicrobial susceptibility testing
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Relevance
o Clinical significance of bacterial isolate
o Predictability of isolate’s susceptibility
o Availability of standardized methods
o Selection of appropriate antimicrobial agents
Accuracy
o Use of reliable methods
o Prompt and thorough review of results
o Prompt resolution of unusual results
Communication
o Augment susceptibility reports with messages that help clarify and explain potential therapeutic problems not necessarily evident by data alone.
6
Q
THE BACTERIA
Bacterial Classification. In this classification, the organisms are divided into 5 kingdoms (Monera, Plantae, Mycetae, Animalia, Protista) and 2 cell types (Prokaryotes, Eukaryotes).
A
Bacteria are neither plants nor animals but they share common characteristics with plants and animals
They belong to the kingdom Monera, and are Prokaryotic according to cell type
Exhibits movement making them similar to animals
Presence of cell wall makes them similar with plants
Differ from Archea due the presence of peptidoglycan layer in bacteria
7
Q
THE BACTERIA: Prokaryotes vs. Eukaryotes Prokaryotes - Without a nucleus - Simpler in structure - Divide by simple binary fission - Ribosome is composed of 50s and 30s subunit
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Eukaryotes
- Has a true nucleus
- Structure is more complex
- Divide by mitosis or meiosis
- Ribosome is composed of 60s and 40s su
8
Q
THE BACTERIA: Prokaryotes vs. Eukaryotes
Clinical Correlation:
The ribosome is unique organelle, which is very important in selective toxicity because having toxicity is one property wherein drugs would be harmful for the bacteria and not for us. Example: the mechanism of action of penicillin: its target is the cell wall and it inhibits cell wall synthesis. Therefore, humans are not adversely affected since we do not have cell
walls.
A
There are also inhibitors of protein synthesis, which affect the ribosomes. These are antibiotics which inhibit the synthesis of 30s subunits (e.g. Aminoglycosides, Tetracyclin), and the 50s subunit (e.g. Chloramphenicol, Macrolide) of ribosomes. Both of which are not present in humans therefore these antibiotics only affect the bacteria.
9
Q
THE BACTERIA: Microbial Dimensions.
Microscopic – requires the use of microscopes to be seen Range: 0.1 nm – 100 μm
Macroscopic – could be seen with the naked eye Range: 1mm and greater
Use Oil immersion objective in viewing bacteria
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Prokaryotic cell external - appendages (flagella, pili, fimbriae) - gycocalyx (capsule, slime layer) cell envelope - cell wall - cell membrane Internal - cytoplasm - ribosomes - inclusions - nucleoid/chromosome - actin cytoskeleton - endospore
10
Q
STAINING
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**The cell wall is the basis why there are gram (+) and (-) bacteria.
11
Q
STAINING: Gram-positive vs Gram-negative Bacteria
Gram (+)
Gram reaction: Remain Violet
Outer membrane: Absent
Porins: Absent
Lipopolysaccharide: None
Teichoic acids: Present
Periplasmic space: Absent
Peptidoglycan layer: Thick (~40 layers) 2 tetrapeptides of adjacent NAM, NAG are linked by peptide bridges
Toxins produced: Exotoxin
Flagellar structure: 2 rings in basal body
Antibiotic resistance: More susceptible
**NAM- N-acetylmuramic acid, NAG- N-acetylglucosamine
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Gram (-)
Gram reaction: Counter-stained red
Outer membrane: Present
Porins: Present
Lipopolysaccharide: Endotoxin, Somatic O Antigen, Core polysaccharide
Teichoic acids: Absent
Periplasmic space: Present
Peptidoglycan layer: Thin (single-layer) 2 tetrapeptides of NAM, NAG are directly linked between D-Alanine and DAG within 2 tetrapeptides
Toxins produced: Endotoxin (Although some have exotoxins)
Flagellar structure: 4 rings in basal body
Antibiotic resistance: Generally more resistant
12
Q
STAINING: Gram-positive vs Gram-negative Bacteria In gram (+) cell walls, there is a compound that could resist decolorization no matter what reagent you add, so it doesn’t change its color, it will still remain as violet. Gram (-) cell walls are lipopolysaccharide-rich, they will be dissolved when you have alcohol (so it will take up whatever stain you put after decolorization)
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- *Cell wall:
- for osmotic protection
- for cell division
- primer for its own biosynthesis
- have sites of antigenic determinants (cell wall itself is antigenic)
- LPS has endotoxin
- Non-selectively permeable
- Gives shape to organisms
- Has diaminopimelic acid which is made up of lysine residues
13
Q
Basic structure of cell wall:
**The peptidoglycan layer is also known as murein or mucopeptide. It is the backbone of organisms that makes up around 50% of gram (+) cell wall, and 5-10% in gram (-).
**How do organisms lose their peptidoglycan? Exposure to UV light or undergo mutation.
Protoplast – Gram positive organism w/o peptidoglycan
Spheroplast –Gram negative organism w/o peptidoglycan
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A complex polymer consisting of three parts: a backbone composed of alternating NAM and NAG; a set of identical tetrapeptide side chains attached to NAM, and a set of identical peptide cross bridges
*The peptidoglycan layer differentiates archae from bacteria. Bacteria have peptidoglycan layer while archae don’t have.
14
Q
Special components of Gram-positive cell walls
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A. Teichoic acids
B. Polysaccharides
15
Q
Special components of Gram-positive cell walls: Teichoic acids
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encompasses all wall, membrane and capsular polymers containing glycerolphosphate or ribitol residues. WTA, LTA, together with peptidoglycan make up
a polyanionic network that provides function relating to elasticity, porosity, tensile strength and electrostatic properties of the envelope.
a. Wall Teichoic Acid (WTA)
b. Membrane/Lipo- Teichoic Acid (LTA)
**Teichoid acid is attached only to the peptidoglycan layer while the lipoteichoic acid is extended up to the lipid bilayer.
16
Q
Special components of Gram-positive cell walls: Polysaccharides
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hydrolysis of gram-positive walls has yielded sugars such as Mannose, arabinose, rhamnose, glucosamine and acidic sugars glucuronic acid and mannuronic acid. It has been proposed that these sugars
exist as subunits of polysaccharides in the cell wall.
17
Q
Special components of Gram-negative cell walls
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(from outer to inner: LPS – Outer membrane - lipoprotein – peptodiglycan – periplasmic space – inner memberane)
18
Q
Special components of Gram-negative cell walls: Porins
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protein channels present in the outer membrane of Gram negative bacteria that permit the passive diffusion of low-MW hydrophilic compounds like sugar, amino acids and ions.
19
Q
Special components of Gram-negative cell walls: Lipopolysaccharide (LPS)
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consists of a complex glycolipid, called Lipid A, to which is attached a polysaccharide made up of a core and series of repeat units. LPS, which is extremely toxic to animals, has been called the endotoxin of gram-negative bacteria because it is firmly bound to the cell surface and is released only when the cells are lysed/disrupted. When LPS is split into lipid A and polysaccharide, all of the toxicity is associated with the former.
20
Q
Special components of Gram-negative cell walls: Lipoprotein
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57 amino acids, stabilizes the outer membrane and anchors it to the peptidoglycan layer
21
Q
Special components of Gram-negative cell walls: Periplasmic space
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contains enzymes break down nontransportable substrates into transportable ones, and detoxifying enzymes (e.g. Beta-Lactamase - hydrolyses the Beta lactam ring present in antibiotics such as Penicillins) – in most books, this is exclusive to gram (-) but in Jawetz, one figure of gram (+) has this feature.
22
Q
Comparison of Endotoxin and Exotoxin.
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Note that endotoxin is found only in gram (-) cell wall. Exotoxin, on the other hand, is found in both but more common in gram (+). Endotoxin is responsible for fever, DIC (Disseminated intravascular coagulation), and shock.
23
Q
- *Clinical Correlation**
- When Beta lactam drugs (penicillin/cephalosporin) enter the porin channel, it will be hydrolyzed/destroyed by beta-lactamase therefore it will not reach the penicillin-binding protein (one mechanism of drug resistance). So we should combine a drug with a beta lactamase inhibitor (eg Coamoxiclav)
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- Pseudomonas has altered porin channels. It is scary because it has marked drug resistance. It is very severe for the patients if there is no drug which is veryeffective.
24
Q
Gram staining Gram (+) Crystal Violet (Primary stain): Violet Iodine (Mordant-chemical that fixes the stain on the bacteria): Violet 95% Alcohol (Decolorizer): Violet Saffranin (Counterstain): Violet
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Gram (-)
Crystal Violet (Primary stain): Violet
Iodine (Mordant-chemical that fixes the stain on the bacteria): Violet
95% Alcohol (Decolorizer): Violet color disappear
Saffranin (Counterstain): Red
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**Clinical Correlation**
- Mycoplasma has no cell wall so penicillin can’t be given to patients with Mycoplasma pneumonia. So we can give macrolide to patients.
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION
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A. Flagella
B. Pili
C. Fimbriae
D. Glycocalyx
D. Ribosomes
E. Endospores
F. Plasmids
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Flagella
- gives an ability to an organism to have run and tumble movement
- Bacterial flagella are thread-like appendages composed entirely of protein, 12-30 nm in diameter. It’s subunits are called Flagellin, which are highly antigenic (H antigen). The flagellum is attached to the bacterial cell body by a complex structure consisting of a hook and a basal body
Lab tests to demonstrate flagella:
- Microscopically: red mounts (stain with carbolfuschin)
- Serologically: H-antigen
- Culture media: if you stabbed it in culture medium, it will diffuse horizontally, so you will see turbidity and horizontal streaks.
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Flagella
Monotrichous – one flagellum located on one end of the bacterial cell.
Amphitrichous- flagella are located at each end of the bacterial cell allowing the bacteria to move from standstill to forward or reverse directly.
Lophotrichous – multiple flagella on one end
Peritrichous- flagella is located all over the perimeter of the cell so the bacteria can easily move in any direction needed
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Pili
shorter and finer than flagella
responsible for twitching surface motility
structure that allows conjugation (transfer of genes between bacteria by direct cell-to-cell contact/bridgelike connections)
Somatic pili – for attachment
Sex/fertility pili – forms a bridge for genetic transfer between 2 organisms (conjugation) – basis for transferring of resistant genes to other organisms
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Fimbriae
Also functions for attachment; usually more attached to the bacterial cell compared to pili which will extend to other organisms
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Glycocalyx
| a glycoprotein-polysaccharide covering that surrounds the cell membranes of some bacteria.
Exists as either:
o Capsule (organized) - one of the mechanisms of immune evasion
-firmly attached
-K antigen
-mucoid appearance of body (those with mucoid colonies have capsules)
-protects bacteria from phagocytosis
-can be visualized using India Ink
o Slime layer (unorganized) - loosely attached
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Ribosomes
Prokaryotic
70s
Subunit :30s + 50s (not mathematically equal to 70s because when they combine, there is a loss of ribosomal mass)
Eukaryotic
80s
Subunit: 60s + 40s (not mathematically equal to 80s because when they combine, there is a loss of ribosomal mass)
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Ribosomes
**Drugs that affect 30s subunit:
Aminoglycosides (e.g. Streptomycin, Amikacin, Gentamicin)
Tetracyclines (e.g. Doxycycline)
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**Drugs that affect 50s subunit:
Chloramphenicol
Macrolides (e.g. Erythromycin)
Lincosamides 9e.g. Clindamycin)
Linezolid
*Mnemonic: buy AT 30, CELL for 50.
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Endospores
One example of an extreme survival strategy employed by
certain low G(+) bacteria is the formation of endospores.
Initiated in response to nutrient deprivation.
Allows the bacterium to produce a dormant and highly resistant cell to preserve the cell's genetic material in times of extreme stress.
Can survive environmental assaults that would normally kill the bacterium.
Important because they are not readily killed by many antimicrobial treatments. A variety of different microorganisms form "spores" or "cysts", but the endospores of low G (+)bacteria are by far the most resistant to harsh conditions.
Spores may be located centrally, subterminally, or terminally.
*2 Types of Endospores emphasized by the lecturer the genus Bacillus and the genus Clostridium
Genus Bacillus is an aerobic endospore
Genus Clostridium is an anaerobic endospore
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PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Plasmids
Small, specialized genetic elements that are capable of replication within at least one prokaryotic cell line
Distinguishing characteristic of prokaryotes is their capacity to exchange small packets of genetic information, carried on plasmids
Extrachromosomal genetic subunits, which are responsible for disease prolongation, recurrence, and resistance.
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Properties of Bacteria Useful for Identification
1. Colonial Morphology
2. Microscopic Morphology
3. Biochemical Test
4. Animal Pathogenicity
5. Immunological Requirements
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Properties of Bacteria Useful for Identification:
| Colonial Morphology
characteristics of the bacteria in culture. Usually the culture is described in terms of shape, color, edge and elevation. It is usually done with unaided eye (not necessarily through microscope).
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Properties of Bacteria Useful for Identification:
| Microscopic Morphology
describes and identifies the culture by using a microscope
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Properties of Bacteria Useful for Identification:
| Biochemical Test
tests that are used to identify Gram Positive and Gram Negative Bacteria. Examples are Catalase Test, Mannitol Salt Agar (MSA), Blood Agar Plates (BAP) for Gram Positive Bacteria and Oxidase Test, Methyl Red/Voges-Proskauer (MR/VP), Simmon’s Citrate Agar
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BIOCHEMICAL TESTS:
1. Blood Agar Plate (BAP)
2. Methyl red/Vogues-Proskauer (MR/VP)
3. Simmon’s Citrate Agar
4. MacConkey Agar
5. Sulfur Indole Motility Medium (SIM)
6. Mannitol Salt Agar (MSA)
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BIOCHEMICAL TESTS: Blood Agar Plate (BAP)
This is a differential medium. It is
a rich, complex medium that contains 5% sheep red blood cells. BAP tests the ability of an organism to produce hemolysins, enzymes that damage/lyse red blood cells (erythrocytes). The degree of hemolysis by these hemolysins is helpful i differentiating members of the genera Staphylococcus, Streptococcus and Enterococcus.
Beta-hemolysis is complete hemolysis. It is characterized by a clear (transparent) zone surrounding the colonies. Staph. aureus, Strep. pyogenes and Strep. agalactiae are b-hemolytic.
Partial hemolysis is termed alpha-hemolysis. Colonies typically are surrounded by a green, opaque zone. Strep. Pneumonia and Strep. mitis are ahemolytic.
If no hemolysis occurs, this is termed gammahemolysis. There are no notable zones around the colonies. Staphylococcus epidermidis is gammahemolytic.
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BIOCHEMICAL TESTS: Methyl red/Vogues-Proskauer (MR/VP)
This test is used to determine which fermentation pathway is used to utilize glucose. In the mixed acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic, succinic, and formic acids). The stable production of enough acid to overcome the phosphate buffer will result in a pH of below 4.4. If the pH indicator (methyl red) is added to an aliquot of the culture broth and the pH is below 4.4, a red color will appear. (Tube on the left). If the MR turns yellow, the pH is above 6.0 and the mixed acid fermentation pathway has not been utilized.
The 2,3 butanediol fermentation pathway will ferment glucose and produce a 2,3 butanediol end product instead of organic acids. In order to test this pathway, an aliquot of the MR/VP culture is removed and anaphthol and KOH are added. They are shaken together vigorously and set aside for about one hour until the results can be read. The Voges Proskauer test detects the presence of acetoin, a precursor of 2,3 butanediol. If the culture is positive for acetoin, it will turn “brownish-red to pink”. If the culture is negative for acetoin, it will turn “brownish-green to yellow”.
**Note: A culture will usually only be positive for one pathway: either MR+ or VP+. Escherichia coli is MR+ and VP-. In contrast, Enterobacter aerogenes and Klebsiella pneumoniae are MR- and VP+. Pseudomonas aeruginosa is a glucose nonfermenter and is thus MR- and VP-
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BIOCHEMICAL TESTS: Simmon’s Citrate Agar
This is a defined medium used to determine if an organism can use citrate as its sole carbon source. It is often used to differentiate between members of Enterobacteriaceae. In organisms capable of utilizing citrate as a carbon source, the enzyme citrase hydrolyzes citrate into oxaoloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO2. If CO2 is produced, it reacts with components of the medium to produce an alkaline compound (e.g. Na2CO3). The alkaline pH turns the pH indicator (bromthymol blue) from green to blue.
Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative.
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BIOCHEMICAL TESTS: MacConkey Agar
This medium is both selective and differential. The selective ingredients are the bile salts and the dye, crystal violet which inhibit the growth of Gram-positive bacteria. The differential ingredient is lactose. Fermentation of this sugar results in an acidic pH and causes the pH indicator, neutral red, to turn a bright pinky-red color. Thus organisms capable of lactose fermentation such as Escherichia coli, form bright pinky-red colonies
MacConkey agar is commonly used to differentiate between the Enterobacteriaceae.
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BIOCHEMICAL Sulfur Indole Motility Medium (SIM)
This is a differential medium. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar (be motile). SIM is commonly used to differentiate members of Enterobacteriaceae. Sulfur can be reduced to H2S (hydrogen sulfide) either by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. If hydrogen sulfide is produced, a black color forms in the medium. Proteus mirabilis is positive for H2S production
Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole. Indole reacts with added Kovac’s reagent to form rosindole dye which is red in color (indole +). Escherichia coli is indole positive.
SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile than the growth will radiate from the stab mark and make the entire tube appear turbid. Pseudomonas aeruginosa and the strain of Proteus mirabilis that we work with are motile.
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BIOCHEMICAL Sulfur Indole Motility Medium (SIM)
Mannitol Salt Agar (MSA)
This type of medium is both selective and differential. The MSA will select for organisms such as Staphylococcus species, which can live in areas of high salt concentration. This is in contrast to Streptococcus species, whose growth is selected against by this high salt agar
The differential ingredient in MSA is the sugar mannitol. Organisms capable of using mannitol as a food source will produce acidic byproducts of fermentation that will lower the pH of the media. The acidity of the media will cause the pH indicator, phenol red, to turn yellow. Staphylococcus aureus is capable of fermenting mannitol while Staphylococcus epidermidis is not
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* *Important Points
1. Do not rely on colonial morphology alone. Do the workup. Do the different tests to identify specific bacteria.
2. All cocci are Gram Positive except Neisseria, Branhamella, Moraxella
3. All bacilli/rod are Gram Negative except genus Bacillus, Clostridium, Listeria, Diphtheria, Actinomycetes, Lactobacillus, Mycobacterium
4. Spiral shaped bacteria that are Gram Negative are Treponema, Bornella and Leptospira
5. Spiral bacteria are viewed using darkfield microscopy
6. Identify bacteria through their shapes. Example: Streptococcus are cocci in chains while Staphylococcus are cocci in clusters
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WAYS TO EXAMINE BACTERIA
A. Unstained
| B. Stained
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WAYS TO EXAMINE BACTERIA: Unstained
1. Wet Mount (e.g. fungi/dermatophytes in KOH)
| 2. Hanging Drop – uses a slide with concavity so that you do not press on the organism
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WAYS TO EXAMINE BACTERIA: Stained
1. Direct (stains the organism)
a. simple
- only uses one reagent
- to determine whether or not there’s an organism
b. differential
- Gram stain or Acid Fast
c. selective
- only stains a specific structure (e.g. spores)
2. Indirect / Negative / Relief (stains the background, making the organism more visible)
- e.g. India ink
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WAYS TO EXAMINE BACTERIA:
| **Culture Media – contains the nutritious environment for the growth of bacteria
A. Physical state
B. Application or function
C. Composition
D. Form or distribution
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WAYS TO EXAMINE BACTERIA: Culture Media
| - Physical state
a. Liquid: broth
b. Solid: agar
c. Semi-solid
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WAYS TO EXAMINE BACTERIA: Culture Media
| - Application or function
a. Simple – nutrient agar (almost all will grow here)
b. Enriched – blood agar (sheep blood is added)
c. Differential – MacConkey agar
d. Selective – MacConkey agar
e. Transport – when you need the sample examined in another laboratory
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BACTERIAL CULTIVATION
A. Lag phase - there is no increase because the bacteria is still trying to find a way to grow (acclimatization or adjustment period)
B. Logarithmic phase or exponential phase - the number of bacteria grows in regular mathematical increments (length of time it takes for bacteria to divide)
C. Stationary phase – the number of cells dying is equal to the number of cells being produced
D. Death or logarithmic decline phase – the culture media nutrients are depleted
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BACTERIAL CULTIVATION: Viable But Not Culturable Bacteria (VBNC)
- Genetic response triggered in starving, stationary
phase cells
- Dormant without changes in morphology (arrested
development)
- Once the appropriate conditions are available (e.g. passage through an animal), VBNC microbes resume growth
- When you want to share very dangerous samples, you should arrest the development, and once it will be used again, it will be inoculated in broth
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BACTERIA LCULTIVATION: Requirements for growth
A. Nutritional
| B. Environmental
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BACTERIAL CULTIVATION: Requirements for growth
| - Nutritional
1. Carbon source – chemolithotrophs (inorganic substrate); heterotrophs (organic carbon)
2. Nitrogen source
3. Sulfur source
4. Phosphorus source
5. Mineral source
6. Growth factors
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BACTERIAL CULTIVATION: Requirements for growth
- Environmental
1. pH
a. Acidophiles (pH 3) (ex: Normal flora of the female genital tract is because of Lactobacillus acidophilus)
b. Alkalophiles (pH 10.5) (ex: Vibrio)
c. Neutrophiles (pH 6-8)
2. Temperature
a. Psychrophilic / cryophilic – 15-20°C
b. Mesophilic – 30-37°C
c. Thermophilic – 50-60°C
d. Hyperthermophilic / thermoduric – >100°C
3. Oxygen requirement / aeration
a. Aerobes – some obligatory aerobes can be better seen in the apical portion of the lungs because there is better aeration
b. Anaerobes
c. Strict/obligate aerobes – will grow only in the presence of oxygen (ex: Clostridium tetany that can only grow in a deep puncture area where there is no oxygen)
d. Strict/obligate anaerobes – will not grow in the presence of oxygen (ex: Mycobacteria: When there are hazy densities in the PA view of an xray (possibly signifying tuberculosis), it is best to take an apicolordotic view to visualize the apices where oxygen is concentrated)
e. Aerotolerant/Facultative – can grow with or without osygen
f. Microaerophilic – needs a small amount of oxygen
g. Capnophilic – needs 5-10% CO2
h. Aerotolerant – fermentative; can use oxygen but is not necessary (they can just tolerate oxygen); without catalase or superoxide dismutase
4. Ionic strength
a. Halophilic - organisms that thrive in high salt concentrations (high ionic strength) (ex Vibrio, all vibrio are halophilic except cholerae and vulnificus. Vibrio can grow on different salt solutions, but at 0% cholerae and vulnificus can still grow)
b. Non-halophilic - low ionic strength
5. Osmotic pressure (Osmophilics can grow at high osmotic pressure)
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PATHOGENESIS OF BACTERIAL INFECTION
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Transmission of infection – mode of transmission; important in preventing the disease
Infectious process – pathophysiology
Genomics and bacterial pathogenicity
Regulation of bacterial virulence factors
Bacterial virulence factors
- Adherence factors (e.g. fimbriae)
- Invasions of host cells and tissues
- Toxins
- Enzyme
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NORMAL FLORA
- Normal inhabitants in a specific site
* Presence of normal flora depends on the organism, specimen, age of patient
* Sometimes an organism in one site is a normal flora there but when it traverses other sites, it will become pathogenic there (ex: Neisseria meningitidis may be a normal flora in the nasopharynx, but it may be pathogenic in the meninges.)
* Some organisms are pathogenic wherever it is found. Ex: Neisseria gonorrhoea is pathogenic wherever it is found.
*Also, the blood is sterile so the presence of organism there is an indication that there is an infection; a stool sample that has no normal flora is a sign of immuno compromised status; an organism is a stool of an adult is normal, but if found in a child there is an infection.
Commensals – maintains the internal milieu
First line of defense against microbial pathogens – prevents opportunistic infections
Assist in digestion
Toxin degradation
Contributes to maturation of the immune system
Shifts in flora – cervical vaginal smears show a shift in flora (from Lactobacillus acidophilus to Gardnerella vaginalis). Epithelial cells are covered with bacilli called clue cells. This is usually seen when someone has multiple sex partners or a new sexual partner
Resident vs. Transient flora: Resident flora are always there. Transient flora change in place.
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Mobile Genetic Elements
Primary mechanisms for exchange of genetic information between bacteria include natural transformation and transmissible mobile genetic elements such as plasmids, transposons, and bacteriophages (often referred to as “phages”).
Transformation occurs when DNA from one organism is released into the environment and is taken up by a different organism that is capable of recognizing and binding DNA. In other cases, the genes that encode many bacterial virulence factors are carried on plasmids, transposons, or phages
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Mobile Genetic Elements
1. Plasmids are extrachromosomal pieces of DNA and are capable of replicating.
2. Transposons are highly mobile segments of DNA that can move from one part of the DNA to another. This can result in recombination between extrachromosomal DNA and the chromosome
3. bacterial viruses or phages are another mechanism by which DNA can be moved from one organism to another. Transfer of these mobile genetic elements between members of one species or, less commonly, between species can result in transfer of virulence factors, including antimicrobial resistance genes.
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REGULATION OF BACTERIAL VIRULENCE FACTORS
Pathogenic bacteria (and other pathogens) have adapted both to saprophytic or free-living states, possibly environments outside of the body, and to the human host. They have evolved complex signal transduction systems to regulate the genes important for virulence
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1. The gene for diphtheria toxin from Corynebacterium diphtheriae is carried on temperate bacteriophages. Toxin is produced only by strains lysogenized by the phages. Toxin production is greatly enhanced when C diphtheriae is grown in a medium with low iron.
2. Expression of virulence genes of B pertussis is enhanced when the bacteria are grown at 37°C and suppressed when they are grown at lower temperatures or in the presence of high concentrations of magnesium sulfate or nicotinic acid.
3. The virulence factors of V cholerae are regulated on multiple levels and by many environmental factors. Expression of the cholera toxin is higher at a pH of 6.0 than at a pH of 8.5 and higher also at 30°C than at 37°C.
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BACTERIAL VIRULENCE FACTORS
| Many factors determine bacterial virulence or the ability to cause infection and disease.
1. Adherence Factors
2. Invasion of Host Cells and Tissues
3. Toxins
4. Enzymes
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BACTERIAL VIRULENCE FACTORS - Adherence Factors
When bacteria enter the body of the host, they must adhere to cells of a tissue surface. If they did not adhere, they would be swept away by mucus and other fluids that bathe the tissue surface.
Bacteria and host cells commonly have net negative surface charges and therefore repulsive electrostatic forces. In general, the more hydrophobic the bacterial cell surface, the greater the adherence to the host cell.
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BACTERIAL VIRULENCE FACTORS - Adherence Factors
Bacteria also have specific surface molecules that interact with host cells. Many bacteria have pili, thick rodlike appendages or fimbriae, shorter “hairlike” structures that extend from the bacterial cell surface and help mediate adherence of the bacteria to host cell surfaces.
For example, some E coli strains have type 1 pili, which adhere to epithelial cell receptors; adherence can be blocked in vitro by addition of d-mannose to the medium.
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BACTERIAL VIRULENCE FACTORS - toxins
| Toxins produced by bacteria are generally classified into two groups: exotoxins and endotoxins
Exotoxins are proteins that are most often excreted from the cell. However some exotoxins accumulate inside the cell and are either injecte directly into the host or are released by cell lysis. Endotoxins are lipid molecules that are components of the bacterial cell membrane.
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BACTERIAL VIRULENCE FACTORS:
toxins - exotoxin
Vaccines have been developed for some of the exotoxin-mediated diseases and continue to be important in the prevention of disease. These vaccines—called toxoids—are made from exotoxins, which are modified so that they are no longer toxic.
Many exotoxins consist of A and B subunits. The B subunit generally mediates adherence of the toxin complex to a host cell and aids entrance of the exotoxin into the host cell. The A subunit provides the toxic activity.
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Exotoxins
1. Excreted by living cell; high concentrations in liquid medium
2. Produced by both gram-positive and gram-negative bacteria
3. Polypeptides with a molecular weight of 10,000–900,000
4. Relatively unstable; toxicity often destroyed rapidly by heating at temperatures above 60°C
5. Highly antigenic; stimulate formation of high-titer antitoxin; antitoxin neutralizes toxin
6. Converted to antigenic, nontoxic toxoids by formalin, acid, heat, and so on; toxoids are used to immunize (eg, tetanus toxoid)
7. Highly toxic; fatal to animals in microgram quantities or less
8. Usually bind to specific receptors on cells
9. Usually do not produce fever in the host
10. Frequently controlled by extrachromosomal genes (eg, plasmids)
Endotoxins
1. Integral part of the cell wall of gram-negative bacteria; released on bacterial death and in part during growth; may not need to be released to have biologic activity
2. Found only in gram-negative bacteria
3. Lipopolysaccharide complexes; lipid A portion probably responsible for toxicity
4. Relatively stable; withstand heating at temperatures above 60°C for hours without loss of toxicity
5. Weakly immunogenic; antibodies are antitoxic and protective; relationship between antibody titers and protection from disease is less clear than with exotoxins
6. Not converted to toxoids
7. Moderately toxic; fatal for animals in tens to hundreds of micrograms
8. Specific receptors not found on cells
9. Usually produce fever in the host by release of interleukin-1 and other mediators
10. Synthesis directed by chromosomal genes
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BACTERIAL VIRULENCE FACTORS:
| toxins - exotoxin
Many exotoxins consist of A and B subunits. The B subunit generally mediates adherence of the toxin complex to a host cell and aids entrance of the exotoxin into the host cell. The A subunit provides the toxic activity.
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BACTERIAL VIRULENCE FACTORS:
toxins - exotoxin (C diphtheriae)
C diphtheriae is a gram-positive rod that can grow on the mucous membranes of the upper respiratory tract or in minor skin wounds. Many factors regulate toxin production; when the availability of inorganic iron is the factor limiting the growth rate, then maximal toxin production occurs. The toxin molecule is secreted as a single polypeptide molecule (molecular weight [MW], 62,000). This native toxin is enzymatically degraded into two fragments, A and B, linked together by a disulfide bond.
Fragment B (MW, 40,700) binds to specific host cell receptors and facilitates the entry of fragment A (MW, 21,150) into the cytoplasm. Fragment A inhibits peptide chain elongation factor EF-2 by catalyzing a reaction that attaches an adenosine diphosphate–ribosyl group to EF-2, yielding an inactive adenosine diphosphate–ribose–EF-2 complex. Arrest of protein synthesis disrupts normal cellular physiologic functions. Diphtheria toxin is very potent.
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BACTERIAL VIRULENCE FACTORS:
toxins - exotoxin (C tetani)
C tetani from the environment contaminates wounds, and the spores germinate in the anaerobic
environment of the devitalized tissue. Infection often is minor and not clinically apparent. The vegetative forms of C tetani produce the toxin tetanospasmin (MW, 150,000) that is cleaved by a bacterial protease into two peptides (MW, 50,000 and 100,000) linked by a disulfide bond. The toxin initially binds to receptors on the presynaptic membranes of motor neurons.
It then migrates by the retrograde axonal
transport system to the cell bodies of these neurons to the spinal cord and brainstem. The toxin diffuses to terminals of inhibitory cells, including both glycinergic interneurons and γ-aminobutyric acid (GABA)–secreting neurons from the brainstem. The toxin degrades synaptobrevin, a protein required for docking of neurotransmitter vesicles on the presynaptic membrane. Release of the inhibitory glycine and GABA is blocked, and the motor neurons are not inhibited
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BACTERIAL VIRULENCE FACTORS:
toxins - exotoxin (C botulinum)
This anaerobic, gram-positive spore-forming organism is found in soil or water and may grow in foods (eg, canned, vacuum packed) if the environment is appropriately anaerobic. An exceedingly potent toxin (the most potent toxin known) is produced. It is heat labile and is destroyed by sufficient heating. There are seven distinct serologic types of toxin. Types A, B, E, and F are most commonly associated with human disease.
The toxi is very similar to tetanus toxin, with a 150,000 MW protein that is cleaved into 100,000-MW and 50,000-MW proteins linked by a disulfide bond. Botulinum toxin is absorbed from the gut and binds to receptors of presynaptic membranes of motor neurons of the peripheral nervous system and cranial nerves. Proteolysis, by the light chain of botulinum toxin, of target proteins in the neurons inhibits the release of acetylcholine at the synapse, resulting in lack of muscle contraction and flaccid paralysis.
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BACTERIAL VIRULENCE FACTORS:
toxins - exotoxin (C perfringens)
Spores of C perfringens are introduced into wounds by contamination with soil or feces. In the presence of necrotic tissue (an anaerobic environment), spores germinate, and vegetative cells can produce several different toxins. Many of these are necrotizing and hemolytic and—together with distention of tissue by gas formed from carbohydrates and interference with blood supply—favor the spread of gas gangrene.
The alpha toxin of C perfringens is a lecithinase that damages cell membranes by splitting lecithin to phosphorylcholine and diglyceride. Theta toxin also has a necrotizing effect. Collagenases and DNAses are produced by clostridiae as well.
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BACTERIAL VIRULENCE FACTORS:
toxins - exotoxin (S aureus)
Some S aureus strains growing on mucous membranes (eg, the vagina in association with menstruation) or in wounds, elaborate toxic shock syndrome toxin-1 (TSST-1), which causes toxic shock syndrome (Chapter 13). The illness is characterized by shock, high fever, and a diffuse red rash that later desquamates; multiple other organ systems are involved as well.
TSST-1 is a super antigen and stimulates
T-cells to produce large amounts of interleukin-2 (IL-2) and tumor necrosis factor (TNF). The major clinical manifestations of the disease appear to be secondary to the effects of the cytokines. Many of the systemic effects of TSST-1 are similar to those of toxicity caused by lipopolysaccharide
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BACTERIAL VIRULENCE FACTORS:
toxins - Lipopolysaccharides of Gram-Negative Bacteria (endotoxin)
The LPS (endotoxin) of gram-negative bacteria are bacterial cell wall components that are often liberated when the bacteria lyse. The substances are heat-stable, have MWs between 3000 and 5000 (lipooligosaccharides, LOS) and several million (lipopolysaccharides) and can be extracted (eg, with phenol-water). The pathophysiologic effects of LPS are similar regardless of their bacterial origin except for those of Bacteroides species, which have a different structure and are less toxic. LPS in the bloodstream is initially bound to circulating proteins, which then interact with receptors on macrophages neutrophils and other cells of the reticuloendothelial system.
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Proinflammatory cytokines such as IL-1, IL-6, IL-8, TNF-α, and other cytokines are released, and the complement and coagulation cascades are activated. The following can be observed clinically or experimentally: fever, leukopenia, and hypoglycemia; hypotension and shock resulting in impaired perfusion of essential organs (eg, brain, heart, kidney); intravascular coagulation; and death from massive organ dysfunction.
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BACTERIAL VIRULENCE FACTORS:
Enzymes
Many species of bacteria produce enzymes that are not intrinsically toxic but do play important roles in the infectious process.
1. Tissue-Degrading Enzymes
| 2. IgA1 Proteases
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The cell membranes of the Archaea differ from those of the Bacteria. Some Archaeal cell membranes contain unique lipids, isoprenoids, rather than fatty acids, linked to glycerol by ether rather than an ester linkage. Some of these lipids have no phosphate groups, and therefore, they are not phospholipids. In other species, the cell membrane is made up of a lipid monolayer consisting of long lipids (about twice as long as a phospholipid) with glycerol ethers at both ends (diglycerol tetraethers).
The molecules orient themselves with the polar glycerol groups on the surfaces and the nonpolar hydrocarbon chain in the interior. These unusual lipids contribute to the ability of many Archaea to grow under environmental conditions such as high salt, low pH, or very high temperature.
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The Peptidoglycan Layer
Peptidoglycan is a complex polymer consisting of three parts: a backbone, composed of alternating N-acetylglucosamine and N-acetylmuramic acid connected by β1→4 linkages; a set of identical tetrapeptide side chains attached to N-acetylmuramic acid; and a set of identical peptide cross-bridges. The backbone is the same in all bacterial species; the tetrapeptide side chains and the peptide cross-bridges vary from species to species, those of Staphylococcus aureus. In many gram-negative cell walls, the cross-bridge consists of a direct peptide linkage between the diaminopimelic acid (DAP) amino group of one side chain and the carboxyl group of the terminal d-alanine of a second side chain.
The tetrapeptide side chains of all species, however, have certain important features in common. Most have l-alanine at position 1 (attached to N-acetylmuramic acid), d-glutamate or substituted d-glutamate at position 2, and d-alanine at position 4. Position 3 is the most variable one: Most gramnegative bacteria have diaminopimelic acid at this position, to which is linked the lipoprotein cell wall component . Gram-positive bacteria usually have l-lysine at position 3; however, some may have diaminopimelic acid or another amino acid at this position.
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The Peptidoglycan Layer
Diaminopimelic acid is a unique element of bacterial
cell walls. It is never found in the cell walls of Archaea or eukaryotes. Diaminopimelic acid is the immediate precursor of lysine in the bacterial biosynthesis of that amino acid. Bacterial mutants that are blocked before diaminopimelic acid in the biosynthetic pathway grow normally when provided with diaminopimelic acid in the medium; when given l-lysine alone, however, they lyse, because they continue to grow but are specifically unable to make new cell wall peptidoglycan.
The fact that all peptidoglycan chains are cross-linked
means that each peptidoglycan layer is a single giant molecule. In gram-positive bacteria, there are as many as 40 sheets of peptidoglycan, comprising up to 50% of the cell wall material; in gram-negative bacteria, there appears to be only one or two sheets, comprising 5–10% of the wall material. Bacteria owe their shapes, which are characteristic of particular species, to their cell wall structure.
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Special Components of Gram-Positive Cell Walls - Teichoic
The term teichoic acids encompasses all wall, membrane, or capsular polymers containing glycerophosphate or ribitol phosphate residues.
These polyalcohols are connected by phosphodiester linkages and usually have other sugars and d-alanine attached. Because they are negatively charged, teichoic acids are partially responsible for the negative charge of the cell surface as a whole. There are two types of teichoic acids: wall teichoic acid (WTA), covalently linked to peptidoglycan, and membrane teichoic acid, covalently linked to membrane glycolipid. Because the latter are intimately associated with lipids, they have been called lipoteichoic acids (LTA).
Together with peptidoglycan, WTA and LTA make up a polyanionic network or matrix that provides functions relating to the elasticity, porosity, tensile strength, and electrostatic properties of the envelope. Although not all gram-positive bacteria have conventional LTA and WTA, those that lack these polymers generally have functionally similar ones.
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Special Components of Gram-Positive Cell Walls - Teichoic
Most teichoic acids contain large amounts of d-alanine, usually attached to position 2 or 3 of glycerol or position 3 or 4 of ribitol. In some of the more complex teichoic acids, however, d-alanine is attached to one of the sugar residues. In addition to d-alanine, other substituents may be attached to the free hydroxyl groups of glycerol and ribitol (eg, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, or succinate).
A given species may have more than one type of
sugar substituent in addition to d-alanine; in such cases, it is not certain whether the different sugars occur on the same or on separate teichoic acid molecules. The composition of the teichoic acid formed by a given bacterial species can vary with the composition of the growth medium.
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Special Components of Gram-Positive Cell Walls - Teichoic
The teichoic acids constitute major surface antigens of those gram-positive species that possess them, and their accessibility to antibodies has been taken as evidence that they lie on the outside surface of the peptidoglycan. Thei activity is often increased, however, by partial digestion of the peptidoglycan; thus, much of the teichoic acid may lie between the cytoplasmic membrane and the peptidoglycan layer, possibly extending upward through pores in the latter
In the pneumococcus (Streptococcus pneumoniae), the teichoic acids bear the antigenic determinants called Forssman antigen. In Streptococcus pyogenes, LTA is associated with the M protein that protrudes from the cell membrane through the peptidoglycan layer. The long M protein molecules together with the LTA form microfibrils that facilitate the attachment of S pyogenes to animal cells.
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Special Components of Gram-Positive Cell Walls - teichuronic acids
The teichuronic acids are similar polymers to techoic acid, but the repeat units include sugar acids (eg, N-acetylmannosuronic or d-glucosuronic acid) instead of phosphoric acids. They are synthesized in place of teichoic acids when phosphate is limiting.
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Special Components of Gram-Positive Cell Walls - Polysaccharides
The hydrolysis of gram-positive walls has yielded, from certain species, neutral sugars such as mannose, arabinose, rhamnose, and glucosamine and acidic sugars such as glucuronic acid and mannuronic acid.
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Special Components of Gram-Negative Cell Walls
Gram-negative cell walls contain three components that lie outside of the peptidoglycan layer: lipoprotein, outer membrane, and lipopolysaccharide
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Special Components of Gram-Negative Cell Walls - Outer membrane
The outer membrane is chemically
distinct from all other biological membranes. It is a bilayered structure; its inner leaflet resembles in composition that of the cell membrane, and its outer leaflet contains a distinctive component, a lipopolysaccharide (LPS)
The ability of the outer membrane to exclude hydrophobic molecules is an unusual feature among biologic membranes and serves to protect the cell (in the case of enteric bacteria) from deleterious substances such as bile salts. Because of its lipid nature, the outer membrane would be expected to exclude hydrophilic molecules as well. However, the outer membrane has special channels, consisting of protein molecules called porins that permit the passive diffusion of low-molecular-weight hydrophilic compounds such as sugars, amino acids, and certain ions. Large antibiotic molecules penetrate the outer membrane relatively slowly, which accounts for the relatively high antibiotic resistance of gram-negative bacteria.
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The Acid-Fast Cell Wall
Some bacteria have cell walls that contain large amounts of waxes, complex branched hydrocarbons (70–90 carbons long) known as mycolic acids. The cell wall is composed of peptidoglycan and an external asymmetric lipid bilayer; the inner leaflet contains mycolic acids linked to an arabinoglycan,
and the outer leaflet contains other extractable lipids. This is a highly ordered lipid bilayer in which proteins are embedded, forming water-filled pores through which nutrients and certain drugs can pass slowly. Some compounds can also penetrate the lipid domains of the cell wall albeit slowly. This hydrophobic structure renders these bacteria resistant to many harsh chemicals, including detergents and strong acids.
If a dye is introduced into these cells by brief heating
or treatment with detergents, it cannot be removed by dilute hydrochloric acid, as in other bacteria. These organisms are therefore called acid fast. The permeability of the cell wall to hydrophilic molecules is 100- to 1000-fold lower than for E coli and may be responsible for the slow growth rate of mycobacteria.
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The mycoplasmas
The mycoplasmas are cell wall-lacking bacteria containing no peptidoglycan. There are also wall-less
Archaea, but they have been less well studied. Genomic analysis places the mycoplasmas close to the gram-positive bacteria from which they may have been derived. Mycoplasmas lack a target for cell wall-inhibiting antimicrobial agents (eg, penicillins and cephalosporins) and are therefore resistant to these drugs. Some, such as Mycoplasma pneumoniae, an agent of pneumonia, contain sterols in their membranes.
The difference between L forms and mycoplasmas is that when the murein is allowed to reform, L forms revert to their original bacteria shape, but mycoplasmas never do.
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Capsule and Glycocalyx
Many bacteria synthesize large amounts of extracellular polymer when growing in their natural environments. With one known exception (the poly-D-glutamic acid capsules of Bacillus anthracis and Bacillus licheniformis), the extracellular material is polysaccharide. The terms capsule and slime layer are frequently used to describe polysaccharide layers; the more inclusive term glycocalyx is also used. Glycocalyx is defined as the polysaccharide-containing material lying outside the cell. A condensed, well-defined layer closely surrounding the cell that excludes particles, such as India ink, is referred to as a capsule. If the glycocalyx is loosely associated with the cell and does not exclude particles, it is referred to as a slime layer.
Extracellular polymer is synthesized by enzymes located at the surface of the bacterial cell. Streptococcus mutans, for example, uses two
enzymes—glucosyl transferase and fructosyl transferase—to synthesize long-chain dextrans (poly-D-glucose) and levans (poly-D-fructose) from sucrose. These polymers are called homopolymers. Polymers containing more than one kind of monosaccharide are called heteropolymers. The capsule contributes to the invasiveness of pathogenic bacteria—encapsulated cells are protected from phagocytosis unless they are coated with anticapsular antibody. The glycocalyx plays a role in the adherence of bacteria to surfaces in their environment, including the cells of plant and animal hosts.
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Endospores
Members of several bacterial genera are capable of forming endospores. The two most common are
gram-positive rods: the obligately aerobic genus Bacillus and the obligately anaerobic genus Clostridium. The other bacteria known to form endospores are Thermoactinomyces, Sporolactobacillus, Sporosarcina, Sporotomaculum, Sporomusa, and Sporohalobacter spp.
These organisms undergo a cycle of differentiation in response to environmental conditions: The process, sporulation, is triggered by near depletion of any of several nutrients (carbon, nitrogen, or phosphorous). Each cell forms a single internal spore that is liberated when the mother cell undergoes autolysis. The spore is a resting cell, highly resistant to desiccation, heat, and chemical agents; when returned to favorable nutritional conditions and activated (see below), the spore germinates to produce a single vegetative cell.
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STAINING
Stains combine chemically with the bacterial protoplasm; if the cell is not already dead, the staining process itself will kill it. The process is thus a drastic one and may produce artifacts. The commonly used stains are salts. Basic stains consist of a colored cation with a colorless anion (eg, methylene blue+ chloride-); acidic stains are the reverse (eg, sodium+ eosinate-). Bacterial cells are rich in nucleic acid, bearing negative charges as phosphate groups. These combine with the positively charged basic dyes.
Acidic dyes do not stain bacterial
cells and hence can be used to stain background material a contrasting color.
The basic dyes stain bacterial cells uniformly unless the cytoplasmic RNA is destroyed first. Special staining techniques can be used, however, to differentiate flagella, capsules, cell walls, cell membranes, granules, nucleoids, and spores.
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The Gram Stain
An important taxonomic characteristic of bacteria is their response to Gram stain. The Gram-staining property appears to be a fundamental one because the Gram reaction is correlated with many other morphologic properties in phylogenetically related forms. An organism that is potentially gram positive may appear so only under a particular set of environmental conditions and in a young culture.
The Gram-staining procedure begins with the application of a basic dye, crystal violet. A solution of iodine is then applied; all bacteria will be stained blue at this point in the procedure. The cells are then treated with alcohol. Gram-positive cells retain the crystal violet–iodine complex, remaining blue; gram-negative cells are completely decolorized by alcohol. As a last step, a counterstain (eg, the red dye safranin) is applied so that the decolorized gram-negative cells will take on a contrasting color; the gram-positive cells now appear purple. The basis of the differential Gram reaction is the structure of the cell wall
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The Acid-Fast Stain
Acid-fast bacteria are those that retain carbolfuchsin (basic fuchsin dissolved in a phenol–alcohol–water mixture) even when decolorized with hydrochloric acid in alcohol. A smear of cells on a slide is flooded with carbolfuchsin and heated on a steam bath. After this, the discolorization with acid-alcohol is carried out, and finally a contrasting (blue or green) counterstain is applied. Acid-fast bacteria (mycobacteria and some of the related actinomycetes) appear red; others take on the color of the counterstain.
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The Flagella Stain
Flagella are too fine (12–30 nm in diameter) to be visible in the light microscope. However, their presence and arrangement can be demonstrated by treating the cells with an unstable colloidal suspension of tannic acid salts, causing a heavy precipitate to form on the cell walls and flagella. In this manner, the apparent diameter of the flagella is increased to such an extent that subsequent staining with basic fuchsin makes the flagella visible in the light microscope.
Negative Staining
This procedure involves staining the background with an acidic dye, leaving the cells contrastingly colorless. The black dye nigrosin is commonly used. This method is used for cells or structures that are difficult to stain directly
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CRITERIA FOR CLASSIFICATION OF BACTERIA
1. Growth on Media
2. Bacterial Microscopy
3. Biochemical Tests
4. Immunologic Tests—Serotypes, Serogroups, and Serovars
5. Genetic Instability
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Selective Media
reside at some sampling sites (eg, the skin, respiratory tract, intestines, vagina), selective media are used to eliminate (or reduce) the large numbers of irrelevant bacteria in these specimens. The basis for selective media is the incorporation of an inhibitory agent that specifically selects against the growth of irrelevant bacteria. Examples of such agents are
* Sodium azide—selects for gram-positive bacteria over gram-negative bacteria
* Bile salts (sodium deoxycholate)—select for gram-negative enteric bacteria and inhibit gram-negative mucosal and most gram-positive bacteria
* Colistin and nalidixic acid—inhibit the growth of many gram-negative bacteria
Examples of selective media are MacConkey agar (contains bile) that selects for the Enterobacteriaceae and CNA blood agar (contains colistin and nalidixic acid) that selects for Staphylococci and Streptococci.
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SOURCES OF METABOLIC ENERGY
Th e three major mechanisms for generating metabolic energy are fermentation, respiration, and photosynthesis. At least one of these mechanisms must be used if an organism is to grow.
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Normal Bacterial Microbiota - skin
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Staphylococcus epidermidis
Staphylococcus aureus (in small numbers)
Micrococcus species
α-Hemolytic and nonhemolytic streptococci (eg, Streptococcus mitis)
Corynebacterium species
Propionibacterium species
Peptostreptococcus species
Acinetobacter species
Small numbers of other organisms (Candida species, Pseudomonas aeruginosa, etc)
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Normal Bacterial Microbiota - Nasopharynx
Any amount of the following: diphtheroids, nonpathogenic Neisseria species, α-hemolytic streptococci; S epidermidis, nonhemolytic streptococci, anaerobes (too many species to list; varying amounts of Prevotella species, anaerobic cocci, Fusobacterium species, etc)
Lesser amounts of the following when accompanied by organisms listed above: yeasts, Haemophilus species, pneumococci, S aureus, gramnegative rods, Neisseria meningitidis
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Normal Bacterial Microbiota - Gastrointestinal tract and rectum
Various Enterobacteriaceae except Salmonella, Shigella, Yersinia, Vibrio, and Campylobacter species
Glucose non-fermenting gram-negative rods Enterococci
α-Hemolytic and nonhemolytic streptococci Diphtheroids
Staphylococcus aureus in small numbers
Yeasts in small numbers
Anaerobes in large numbers
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Normal Bacterial Microbiota - Genitalia
Any amount of the following: Corynebacterium species, Lactobacillus species, α-hemolytic and nonhemolytic streptococci, nonpathogenic Neisseria species
The following when mixed and not predominant: enterococci, Enterobacteriaceae and other gram-negative rods, Staphylococcus epidermidis, Candida albicans, and other yeasts
Anaerobes (too many to list); the following may be important when in pure growth or clearly predominant: Prevotella, Clostridium, and Peptostreptococcus species
