all of the chemical processes that take place in a cell
process by which a cell takes materials from the environment/catablic reactions (simple nutrients) and converts them to the various components of the cell
Typically, the more nutrients there are readily available, the higher the growth rate of bacterial colonies.
process by which various chemicals are broken down, releasing energy. The cell uses this energy to drive other processes (e.g. anabolism)
Conservation of energy from chemical reactions involoves:
What kind of bonds are formed during an oxidative reduction reaction?
Polar covalent bonds
ionic bons (e.g. Na+ + Cl- → NaCl)
In any case, redox reactions involve a shift in the balance of electrons
In a redox reaction, the e- donor becomes ___ and the acceptor becomes ____.
A totally reduced carbon is bound to ____ and a totally oxidized carbon is bound to ____.
Likewise, a totally reduced oxygen is bound to ____.
4 hydrogens (CH4)
2 oxygens (CO2)
2 hydrogens (water)
Are lipids reduced or oxidized? How does this make them useful?
They are very reduced. They contain a lot of energy due to the amount of times they can be oxidized as they are converted to H2O and CO2
Air in the atmosphere normally contains what percent of O2 and CO2?
cannot grow in the presence of air/molecular O2 (due to a sensitivity to the presence of oxygen)
are able to grow in the absence of O2
do they undergo fermentation?
Grow where in thioglycollate broth?
require presence of O2 for energy metabolism;
do not undergo fermentation
-grow at the top of the broth when cultured
can form toxic superoxide radicals (O2•-)(has one unpaired electron, making it very reactive)
How do aerobic bacteria survive in the presence of O2?
They have enough superoxide dismutase (SOD) and catalase to remove toxic metabolites that result from the incomplete reduction of molecular oxygen
Two types of obligate anaerobes
Strict- grow in environments containing no more than 0.5% O2. Can be killed quickly with exposure to air.
Moderate- most clinically relevant anaerobes. Will grow in environments 2-8% O2
-grow in presence of O2?
-where do they grow in thioglycollate broth?
Can grow O2 presence, but grow best in an anaerobic environment
-have enough SOD, so they can grow anywhere in the broth (uniform amount throughout broth)
-can grow in the presence/absence of O2?
-have suffiecient ___ and ____
-grow best in presence or without O2? Why
-Tend to grow where in Tho\ioglycollate broth?
-Can grow in the presence or absence of O2
-They have sufficient SOD and catalase/peroxidase
-Grow best in presence of O2 bc they use it in energy metabolism
-tend to grow more near the top, but can grow anywhere in the broth
Facultative anaerobes can change thier ___ to cope with environmental changes.
Obligate anaerobes lack
where do they grow in thioglycollate broth?
catalase and peroxidase or have such small amounts that they die from toxic effect of H2O2
tend to grow at bottom of broth
E. Coli (Escherichia Coli... spelling?)
faculatative anaerobe, highly versatile
allows culture of obligate aerobes(top), obligate anaerobes(bottom), facultative anaerobes(tend to be more @top), aerotolerant anaerobes (all over), and microaerophiles (will grow in the middle below the surface)
Three growth modes for E. coli
fermentation (mixed acid fermentation)
Strict anaerobes lack:
What does this enzyme do?
Superoxide dismutase (SOD)
Converts a superoxide radical (O2•-) to peroxide (H2O2)
Note: H2O2 is still toxic
converts peroxide (H2O2) to nontoxic molecules
relatively low molecular weight, ferric ion specific chelating agents elaborated by bacteria and fungi growing under low iron stress
-take iron (from hosts) and concentrate it
important to the functioning of many enzymes, epecially the electron transport chain
where is iron found in mamalian tissues? (4 examples)
tightly bound in a variety of proteins: iron is pretty toxic if left by itself
heme- prosthetic group for hemoglobin, myoglobin, and several enzymes
ferratin- inside cells
transferrin- for iron transport between tissues
lactoferrin- in milk, tears, mucosal secretions, and granules of PMNs
Where/How do bacteria typically break down macromolecules for nutrients?
They use enzymes to break down large molecules outside of the cell membrane, then bring in the smaller subunits using active or passive transport
3 carbon sugar, provides carbon skeleton
converging point for many pathways
3 pathways bacteria us to convert glucose to pyruvic adic
The carbons from pyruvic acid can be: (2)
- further catabolized for energy production
- used in anabolic reactions in the synthesis of new amino acids, lipids, carbs, or precursors for nucleic acids
How is energy captured in the catabolism of glucose
formation of high energy molecules (ATP) or reduced coenzymes
What is pyruvate used for? (2)
provide the intermediate that enters into tricarboxylic acid (TCA) or Krebs cycle
what are reduced conenzymes used for?
form proton gradient across cell via aerobic/anaerobic respiration
metabolic process that involves electron transport along a series of membrane associated electron carriers to a final inorganic electron acceptor
when the final inorganic electron acceptor is something other than O2
e.g. NO3-2, SO4-2, CO3-2
final inorganic electron acceptor is O2
an organic molecule (often pyruvate) is the terminal electron acceptor. ETC is not used (less energy produced)
The Entner-Doudoroff Pathway is used instead of ____ in some bacteria.
Examples of bacteria that do this: (2)
Pseudomonas aeruginosa (gram -)
Enterococcus faecalis (gram +)
The reduced coenzymes from the TCA cycle are reoxidized via the ____.
Electron Transport chain
Where does it occur?
Energy input required?
reaction occurs in both aerboic and nonaerobic conditions
Needs investment of 2 ATP
Net products of glycolysis (per mole of glucose)
2 moles of pyruvate
2 moles of ATP (via chemical/ substrate level phosphorylation)
2 moles of NADH
To continue glycolysis, the cell needs a way to ____. How does this happen?
via repiration or fermentation
The pentose phosphate pathway is important in : (2)
Can it yield any ATP?
biosynthesis (Source of NADPH) and catabolism (source of 5-carbon sugars for nucleic acids)
Yields 1 net ATP if used for energy
Entner-Doudoroff pathway is only seen in?
Products per mole of glucose?
Most bacteria that use this pathway are aerobic/anaerobic?
prokaryotes (that may lack an essental glycolysis enzyme)
1 NADH, 1 NADPH, 1 pyruvate, and 1 ATP net per mole of glucose
pyruvate or pyrubate derivative serves as the termianl electron acceptor
ETC is not used
uses membrane embedded electron transport chain and usually an inorganic electron acceptor
The TCA cycle is for
Energy production (complete oxidation of pyruvate) to CO2
Biosynthetic Function- provides carbon skeletons for use in biosynthesis
In the presence of an inorganic electron acceptor (O2 or NO3-2), the NADH from glycolysis, and the NADH & FADH2 from the TCA cycle are ____.
oxidized via the electron transport chain
What type of respiration uses O2 as the final electron acceptor and creates a H+ gradient across the cell membrane?
NO3-2, SO4-2, or CO3-2 used as terminal electron acceptor.
Less H+ gradient produced, and lower ATPs
Bacterial cells replicate using
the phase when bacteria are first added to a new medium and they require time to adapt their metabolism
No increase in cell numbers in this phase
nutrients begin to be used by bacterial cells and cell growth is exponential
cell growth slows due to lack of nutrients and buildup of toxic materials
same # of cells duplicating and dying
phase of decline/ death phase
exponential death of bacteria due to a buildup of toxic material and no more resources
Not as exponential as the log phase
*do not renew/add anything to the medium during the study of population dynamics of a batch culture
species found in the upper respiratory tract and skin of all warm blooded animals
What are the cell wall virulence factors of Staphylococcus aureus?
-peptidoglycan and teichoic acids may have endotoxin like activity (stimulate WBC to release cytokines)
-modified penicillin-binding protein provides resistance to penicillins and cephaloporins
a concave glass/quartz lens refracts light and is used for magnification of an image at a distance away from the focal point
image is reversed and upside down
Protein virulence factors of S. aureus?
-surface proteins that act as adhesins
-surface proteins that subert the immune system
-secreted toxins (cytotoxic alpha-toxin, toxic shock syndrom toxin-1, enterotoxins, exfliative toxins, and panton-Velentine leukocidin (kills WBCs)
-secreted enzymes- promote bacterial spreading through tissues due to tissue destruction
What regulates the expression of various S. aureus genes? (type of genes)
What is an example of one of these?
Global regulatory genes
The best studied one is a quorum sensing regulatory system that upregulates the expression of surface proteins
-enables the bacteria to optimize virulence gene expression and host colonization
surface and secreted proteins in S. aureus are _____ phase dependent.
Methods to identify microbial agents of disease (4)
-direct microscopic visualization
-culitvation and identification of the organism
-serological and cytological methods (detect microbial antigens, detect the hosts immune response)
-detection of microbial DNA or RNA
What color is the shortest visible light wavelength and what is the resolution limit for a light microscopy
blue wavelength is the shortest wavelength and it gives us a resolution limit of ~200nm with light microscopy
Shorter wavelengths give ___ resolution
abitlity to distinguish two closely placed points
UV light has a shorter or longer wavelength than visible light?
brightfield (light) microscopes
field of view is bright if:
Light source is under specimen and light passes through the specimen to then enter into the objective lens
brightfield (light) microscopes
usually 4x, 10x, 40x, or 100x power
The 100x needs immersion oil in order to not lose light
brightfield (light) microscopes
immersion oil has the same refractive index as the glass of slide and objective lens T/F
What is the function of immersion oil
allows more light to enter into the small diameter, high magnification (90x-100x) lens
brightfield (light) microscopes
-the microscope has how many ocular lenses?
-Total maginificaiton = ?
-magnification of objective lens x magnification of occular lens
*most modern microscopes also have a condensor lens to direct light through specimen
On light microscopes, why do objective lenses have different lengths?
Each lens set has a different focal lengths
Uses the same objective and occular lenses as brightfield microscope T/F
A special condensor only allows ____ light to reach the specimen.
This causes the specimen to appear____ against a ___ background.
oblique, scattered light
Obect looks bright against a dark background
20nm (10x more resolution than brightfield)
Phase contrast microscopy creates what kind of image?
It enables visualization of:
enables visualization of the internal structures of unstained cells
differential interference contrast microscopy (DIC) is a techique used to enhance:
contrast in unstained, transparent samples
flurochromes (used in flurorescent microscopy) absorb what kind of light?
Then they emit:
short wavelength UV light
Then they emit energy at a higher visible wavelength
employs a fluorescent dye linked to an antibody. The antibody directly binds with the target antigen
tagged antibody is a second antibody. It binds to another antibody that actually binds the target antigen
Transmission electron microscopy
electrons are fired through specimen and create an image on a detector screen.
Transmission electron microscopy
How must specimen be prepared?
what is the max resolution compared to LM?
ultrathin sectioning and staining with heavy metals such as osnium, uranium, or lead for increased contrast
Max resolution: 1000x greater than LM
Scanning electron microscopy
surface of specimen is coated with heavy metal and electron beam scans across its suface, rather than passing through.
reflected electrons are collected and form a 3D like image on a high resolution cathode-ray tube
specimen is placed on a slide and mixed with a salt solution
to detect fungi in tissue scrapings (using wet mount technique), what solution should be used?
10% KOH solution and a stain. The KOH solution will dissolve mammalian and non cellular material, leaving the reistant fungal cell walls.
What can india Ink used for?
India ink can be used to visualize the thick capsule of Cryptococcus neoformins in freshly obtained cerebrospinal fluid
Methods to phentypically classify bacteria
Morphology (cell shape, cell arragements, staining characteristics)
Basic dyes have what kind of charge? This means they bind easily to __.
examples of basic dyes (5)
They bind easily to negatively charged molecules, such as nucleic acids, many proteins, and surfaces of bacterial cells
Acid dyes have what kind of charge? Are they used for bacteria typically?
negatively charged/ are not typically used for bacteria
bind to positively charged cell structures
Simple stains are used for
to tell cell shape, size, and cell arrangements
Differential stains are used for:
Examples of differential stains:
divide bacteria into seperate groups based on staining properties
e.g. gram stain, acid fast stain
Gram staining process (6 steps)
1. Heat fix specimen to slide & flood w crystal violet solution. Leave for 1 min
2. rinse the slide, then flood with iodine solution. Leave for 1 min
3. Decolorize w acetone for 5 seconds (not too vigorously tho)
4. wash slide in water immediately. Gram neg bacteria wont be visible now
5. apply safranin counterstain for 30 seconds
6. wash with water, blot, and air dry.
Violet colored bacteria: gram pos
Red colored bacteria: gram neg
During the streak plate technique, it is important to __ the loop between successive regions
How to fix bacteria to a slide?
spread culture in thin film over slide, let air dry, then pass slide through flame to fix it.
(see slide 69)
Acid fast stain used to stain (what organisms?)
What color will they stain? What is used as the counter stain?
Acid fast stains used for staining genuses Mycobacterium and Nocardia and oocyts of certain protozoa
They stain red.
Methylene blue is used as counter stain. All other cells will appear blue
Ziehl-Neelson Stain (same thing as acid fast) (Kinyoun modification)
Organism is suspended in saline soluntion.
Slide is flooded with carbol fuchsin and phenol (3 min), then rinsed w H2O
Slide decolorized using 3% HCL in 70% water (~2 min)
rinse with water, then slide flooded with methylene blue counterstain (30 sec)
rinse w water and air dry
In acid fast staining you can the blue stained cells are always gram positive T/F
every cell besides acid fast bacteria (which will stain red) will appear blue. This includes all gram pos, gram neg bacteria and eukaryotes.
What stain procedure can be used to visualize Klebsiella pneumoniae?
simple stain to stain the cells and a negative stain to demonstrate their capsule
Negative (capsule) staining
stains the background and shows true cell size and can demonstrate capsules
allows you to see a green endospore and red vegatative cell
Stains volutin (polyphosphate) in Corynebacterium diptheriae
Are endospores easily stained?
What are two species of bateria that form endospores that we are concerned with?
Baccillus and Clostridium
What compound do you use to stain an endospore?
where is this used?
staining in a different color than that of the dye
Can be use to detect volutin (black) granules within green Corunebacterium cells) (using Albert's stain)
Why stain bacterial flagella?
flagella are too slender to be seen with LM, so flagellar stain coats can make them more apparent. Not routinely done, but can provide important taxonomy info.