Bacteriology (trans 4) Flashcards
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
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.)
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
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
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
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.
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
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.
INOCULATION, INTUBATION, AND ISOLATION - Effective antimicrobial susceptibility testing
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.
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).
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
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
Eukaryotes
- Has a true nucleus
- Structure is more complex
- Divide by mitosis or meiosis
- Ribosome is composed of 60s and 40s su
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.
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.
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
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
STAINING
**The cell wall is the basis why there are gram (+) and (-) bacteria.
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
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
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)
- *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
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
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.
Special components of Gram-positive cell walls
A. Teichoic acids
B. Polysaccharides
Special components of Gram-positive cell walls: Teichoic acids
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.
Special components of Gram-positive cell walls: Polysaccharides
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.
Special components of Gram-negative cell walls
(from outer to inner: LPS – Outer membrane - lipoprotein – peptodiglycan – periplasmic space – inner memberane)
Special components of Gram-negative cell walls: Porins
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.
Special components of Gram-negative cell walls: Lipopolysaccharide (LPS)
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.
Special components of Gram-negative cell walls: Lipoprotein
57 amino acids, stabilizes the outer membrane and anchors it to the peptidoglycan layer
Special components of Gram-negative cell walls: Periplasmic space
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.
Comparison of Endotoxin and Exotoxin.
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.
- *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)
- 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.
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
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
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.
PROPERTIES OF BACTERIA USEFUL FOR INFORMATION
A. Flagella B. Pili C. Fimbriae D. Glycocalyx D. Ribosomes E. Endospores F. Plasmids
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.
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
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
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
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
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)
PROPERTIES OF BACTERIA USEFUL FOR INFORMATION: Ribosomes
**Drugs that affect 30s subunit:
Aminoglycosides (e.g. Streptomycin, Amikacin, Gentamicin)
Tetracyclines (e.g. Doxycycline)
**Drugs that affect 50s subunit: Chloramphenicol Macrolides (e.g. Erythromycin) Lincosamides 9e.g. Clindamycin) Linezolid *Mnemonic: buy AT 30, CELL for 50.
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
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.
Properties of Bacteria Useful for Identification
- Colonial Morphology
- Microscopic Morphology
- Biochemical Test
- Animal Pathogenicity
- Immunological Requirements
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).
Properties of Bacteria Useful for Identification:
Microscopic Morphology
describes and identifies the culture by using a microscope
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
BIOCHEMICAL TESTS:
- Blood Agar Plate (BAP)
- Methyl red/Vogues-Proskauer (MR/VP)
- Simmon’s Citrate Agar
- MacConkey Agar
- Sulfur Indole Motility Medium (SIM)
- Mannitol Salt Agar (MSA)
