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Why classify organisms?
- Establish similarities
- Can influence therapy
- Predict common characteristics/features (growth, habits)
- To place in appropriate group which allows you to predict characteristics of organisms
- Common language
- facilitates the retrieval f information and appropriate study
- Living Organisms
- Asses viability
- Methalin Blue Yeast Viability
- Trypan blue exclusion
Methalin Blue Yeast Viability
- Specimen w/ Cata Albacins and stain w/ methalin blue and look for yeast cells.
- Dead cells = blue
- Live Cells = clear
- Hep. Vaccine is made in yeast
Trypan Blue Exclusion
- Used to test viability of bone marrow donations.
- Get membrane of bone marrow, add trypan blue.
- Viable Cell = Will not accumulate trypan blue
- Dead Cell = Stains blue
Cell Viability and Viruses
- Relevant in virus detentions because the virus is associated with characteristics cytopathogenic effects such as cell death and rounding.
- If cells are dead = cannot tell if virus or not so you have to show viability.
- Any change seen after is due to the virus cuz the virus killed the cell and that is how you can infer it.
Basic principles of Microscopy
- ID organisms and diagnose infections
- Formula: R=lamda/2NA
- Most useful unit: micrometer = 10^6
- Deals with resolution
- - Lamda = wavelength
- - NA = numeric aperture
- - Smaller R is a better value
- Smaller wavelength = better resolution
Types of Microscopes Used
- Unaided Human Eye
- Compound Light Microscope
- Scanning electron microscope
- Transmission electron microscope
- Scanning tunneling microscope
- Atomic Force Microscope
Compound Light Microscope
200 nm-10 mm
Scanning Electron Microscope
0.4 nm - 1mm
Transmission Electron Microscope
0.078 nm - 100 µm
Scanning tunneling Microscope
0.01 nm - 10 nm
Atomic Force Microscope
1 nm - 10 nm
Benefits of Immersion Oil
- Cannot see in focus unless you have immersion oil because it has the same refractive index as glass and it actually extends the objective lens (focus light) and it has to have contact with the oil.
- Basically you can see better because it acts as another lens
Used for specific antibodies not useful clinically
- Indicates specific characteristics of organisms
- More on notes, listen to voice
- Domain - bacteria, archaea, eukarya
- Kingdom - Animalia, Plantae, fungi
Taxonomic Kingdom Categories
Linnaus and Taxonomic Categories
- Linnaus's goal was to classify organisms to catalog them.
- Modern goal is to understand relationships among groups of organisms
- - Reflect phylogenetic hierarchy
- - Emphasis on camparison of organisms genetic material
- - Led to proposal to add domain
Taxonomic ID Classifications
- Physical characterisitcs
- Biochemical tests
- Serological tests
- Phage typing
- Analysis of nucleic acids
- Antibody - antigen reactions
- Antibodies found in blood of animals that are raised with particular anitbody
- Inc of antibody content is typically indicative of infection.
- Can look at relative concentrations of IGG vs IGM
IGG vs IGM in Serological Classifications
- IGG = the longest half life and has the highest concentrations.
- Think of below as a point on a graph:
- During an infectious process the first antibody produced is IGM and the second AB produced is IGG. So if a patients blood has more IGG than IGM it means that it's an old infection. If there is more IGM than IGG it means that is a recent infection.
- Antibody levels document response to to vaccination and to confirm protection.
- To do this you measure the Titer.
- All about concentration ratios.
- A way of expressing concentrations. To get titer you need to use serial dilution.
- Measures antibody levels to determine if you have contracted an infection
- Bacterial virus
- Also have plaques
- Specific to each strain
- Can tell because it kills bacteria and you get lyses.
Plaques in Phages
Absence of bacteria and where ever the virus infects bacteria you get the holes which are plaques
Basis of Phages
- Phage typing enables the the indicative/definitive ID of different strains of bacteria that establish the origin or the source of infection.
- Identical strains are usually infected by the same phage pattern
Uses of Phages
- You have 3 patients: S. Aureus for example.
- Patient A has a phage pattern of 1, 8, 16, and 32.
- Patient B has a phage pattern of 1, 12, 24, and 68.
- Patient C has a phage pattern of 2, 4, 10, and 12
- Doctor has a phage pattern of 2, 4, 10, and 12.
- Q. Where did the S. Aureus come from.source?
- A. The source is the doctor because the phage patterns match
- Consist of dichotomous keys
- In the biochemical unknown you have either gram + or gram - and in morphology it could be a rod or coccus
- Shows characteristics of organisms so if you look at the biochemical profile you will end up with the definitive ID for the species.
- Think of this as a flow chart we do in lab to ID the organisms, it's either this or that and you follow steps
What is the difference between citrobacter and enterobacter
Citrobacter doesn't produce acetoin
T/F. Humans are capable of anerobic respiration?
Detects acetoin and butanediol
Detects mixed acid fermentation
- Total of all biochemical reactions in an organism.
- Basic feature of Life
- Consists of anabolic and catabolic reactions
- Synthetic reaction.
- Makes new macro molecules for cells.
- Linked with ATP production/use
- Breaks down macro molecules to release energy as ATP
- Link by ATP production/use
Why is Metabolism Clinically Relavent. 2 Examples
- 1. Infection associated with distinctive odors produced by volatile end products of microbial metabolism.
- - Whiff test for G. Vaginalis
- 2. Lactate relevance in sepsis and septic shock
Whiff Test for G. Vaginalis procedure
- Add potassium hydroxide to cervical scraping slide, then you will get a distinctive fishy amine odor which confirms diagnosis of g. Vaginalis
- #1 of why Metabolism is relevant cuz of odor
- Unwind DNA in order to replicate.
- Can be in bacterial form or in eukaryotic form that is found in humans
Antibody Level Applications With Titer: Single Static Titer USE IGG VS IGM FOR SEROLOGY RELAVENCE
Single Static Titer: You can determine if a patient is infected by taking the ratio of titer of the patient vs the gen. population titer. If the gen. population has a ratio of 1:16 and the patient has a ratio of 1:32 that means that the patient is infected because the antibody levels are 1 fold higher.
- Means that you have a high concentration with the most antibodies. So you have to dilute that more.
- Think of it as 10 spoons of sugar and 2 spoons of sugar in different cups. 10 spoons will be sweeter so you have to put more water to make it normal.
Definitive ID: Biochemical Profile
- Part of metabolism
- Bacteria ID by biochemical profile (end products).
- Enzymes Present
- Substrates Utilized
E. Coli Vs. Enterobacter part of Biochemical Profile
- They are differentiated by IMVIC tests
- E. Coli: Positive for Indole and Methyl Red. Negative for Vp and Citrate
- Enterobacter: Positive for VP and citrate, negative for indole and methul red
Volatile End Products
- Part of Metabolism
- C. Defficile ID is based on production of isocaproic acid in feces.
- Anaerobes ID by gas chromatography detection of end products
Methods of Control
- Physical/chemical methods use of heat/others
- Depend on inhibiting key enzymes, protein denaturation, inhibition of metabolic pathways by antibiotics
- Infectious diseases associated with disturbances in homeostasis and metabolic activity.
- Sepsis and septic shock. Arterial blood gases, vital signs, blood ph, other
Stages of Metabolism
- Krebs Cycle
- Oxidative Phosphorylation
- Follows the order above one happens after the other
- Produces NADH and FADH, ATP, CO2
- Occurs with aerobic and anaerobic respiration not with fermintation
- Involves and Electron Transport Chain
- Involved in Aerobic and Anaerobic Respiration
- Read about electron transport chain to understand
What are enzymes?
Substance produced by a living organism that acts as a catalyst to bring about specific biochemical reactions
Clinical Applications of Enzyme Activity
- Fluodroquinolones and bacterial DNA gyrase
- Sulfur Drugs as antibiotics, will deal with specificity of action
Action of Fluodroquinolones (FQ) on Bacterial Gyrase
- FQ's (antibiotic)
- Will have more affinity on the bacterial gyrase and very little in eukaryote gyrase.
- - It will inhibit bacteria in the bacterial gyrase which is based on affinity (more binding capabilities)
- FQ prefers to bind to bacterial gyrase because of the affinity. Specifically inhibit bacteria replication which is the goal
- Specificity relies on the difference in the pathways of enzymes used to make folic acid.
- When treating with sulfur drugs it will inhibit bacterial pathways cuz of the different enzymes and the sulfur drugs actually PABA which mimics folic acid synthesis.
- So if you unhibit it that means the folic acid stops and tha prevents DNA synthesis and as a result the bacteria are inhibited
Six Categories of Enzymes
- Ligases or polymerases
- Lower activation energy
- - makes reactions easier to achieve so reaction is faster
- Measured in how fast reactions occur
- Provides a physical site for the collision of reactions
Things that influence Enzymes
- Enzyme substrate concentrations
- Presence of inhibitors
Things that inhibit Enzymes
- Substances that block enzyme active site
- Do not denature enzymes
- Three types
How Enzymes are Inhibited
The reaction rate slows down or terminates activity
Three types of Enzyme Inhibitors
- Competitive inhibitor
- Allosteric inhibition
- Feedback Inhibition
Inhibit by competing with substrate and you can overcome it by inc. substrate concentration
- Could be activator or inhibitor.
- Inhibitions occurs when the allosteric inhibitor binds to an active site of an enzyme which changes the active site to an inactive site because it changes shape of the binding site and makes it inactive
- Org. needs an end product.
- Shuts down when there is enough and when decreases it starts again.
- Initial reactions Energy-
- Energy-Investment Stage
- - Spend money to make money
- Lysis Stage
- - Destruction on voice
- Energy-conserving stage
- - Recover ATP
Why is lactate relevant in sepsis and septic shock?
- #2 Of why metabolism is relevant.
- Critical in diagnosis of cryptic septic shock
- Relevant because during sepsis and septic shock you have ischemia, so you diminish normal metabolic activity since you lack oxygen. Because of that you get lactate produced at much higher concentrations than normal as a by product of fermentation
Are autocatalytic they are the exception and are not proteins like other enzymes
Michalis Menten Plot
- Km = 1/2 of Vmax and is a measure
- Vmax = Substrate concentrations where reactions are fastest/highest possible. (where the graph curves)
- Vmax is achieved with all active sites are saturated with substrate
- Km = indicative of the affinity of substrate for the active site of the enzyme
- Read in notes and listen to lecture
- Breaks down glucose to form pyruvates
- Produces 4 ATP and 2 NADH, but uses 2 ATP for the proccess and nets 2 ATP and 2NADH
- Converts exogonic reaction to endogonic reaction
- Step 2 of Glycolysis
- Breaks down glucose to form pyruvates
Energy Conserving Stage
- Stage 3 of Glycolysis
- Energy Recovery Stage
- Converts pyruvate to make ATP and NADH
Energy Investment Stage
- Stage 1 of Glycolysis
- Spend money to make money into pyruvate
Pyruvate Conversion Possibilities
Can be fermented, can go through oxidative phosphorylation when oxygen is present in aerobic respiration, or can be broken down anerobically using alternate acceptors
Useful as an adjunct to treating flesh eating disease, dicubibus ulcers, gangrene, and wounds that do not heal.
Why Anaerobes and Anaerobes
They cannot survive with oxygen because they don't have the oxygen protective enzymes
Alternate Anaerobic Acceptors
Sulfate, nitrate, carbonate, and thiosulfate
Glycolysis and Krebs Cycle
- Are major amphibolic pathways. Function in both anabolic and catabolic activity
- All major macro molecules in any org. are cycled, synthesized, and broken down through Krebs and Glycolysis
Largest # of bacteria per gram of fecal matter is?
Krebs Cycle Produces
- 2 molecules of ATP
- 2 molecules of FADH
- 6 molecules of NADH
- 4 molecules of CO2
- FADH and NADH do not produce ATP until reaching the electron transport chain
Electron Transport Chain
Most significant ATP production occurs here
What is involved in having a successful Electron Transport Chain
Need to have an intact membrane, electron carriers and proteins that flow out
- Only way that the protons can get back into the membrane of the electron transport chain.
- Uses chemiosmosis to work which has 3 parts loose, tight, and open
- Protons pumped out of electron transport chain.
- Then Protons build up outside of membrane
- Then protons enter membrane through ATP Synthase which then follows below steps
- Loose = accepts things
- Tight = Groups together
- Open = Releases back into electron transport chain
T/F. Life and death can be defined as metabolism
T/F. Life is a tiny electron current that is directed by the sun.
Proton Motive Force (PMF)
Force implies potential energy due to electron and proton flow
Oxygen serves as final electron acceptor
Molecule other than oxygen serves as final electron acceptor
Question? An aerobic org. was fed radioactive labeled oxygen where does this oxygen end up?
Question? A fuel tank was painted with cyanide paint to prevent microbial growth, 2 days after the bacteria were recovered from the tank. What process was being used to produce ATP?
Alternatives to Glycolysis
- Pentose phosphate pathway
- Entner-Doudoroff pathway
- Uses organic molecule within cell as final electron acceptor
- Sometimes cells cannot completely oxidize glucose by cellular respiration
- This provides the cells with source of NAD
Similarities between Aerobic, Anaerobic Resp. and Fermentation
- All 3 process are common to glycolysis
- All produce ATP substrate level phosphorylation
Differences between Aerobic, Anaerobic Resp. and Fermentation
- Different end products are produced
- Aerobic = most efficient
- Aerobic & Anaerobic = Role of CO and cyanide
- Aerobic & Anaerobic = Include Oxidative phosphorylation
- Aerobic & Anaerobic = Has electron transport chain