Final

Probe Microscope

- A minuscule, pointed electronic probe to magnify more than 100,000,000x. - Two types: 1. SCANNING TUNNELING MICROSCOPE (STM) 2. ATOMIC FORCE MICROSCOPE

Flagellar Stain

- Bacterial flagella are extremely thin and thus normally invisible with light microscopy, but their presence, number and arrangement are important in identifying some SPECIES, including some PATHOGENS. - FLAGELLAR STAINS, such as PARAROSANILINE and CARBOLFUCHSIN, in comnination with MORDANTS - chemicals that combine with a dye and make it less soluble and therefore affix it in a material - are applied in a series of steps. - Flagellar stains bind to flagella, increasing their diameter and colorizing them, which increases contrast and makes the flagella visible.

Cell Theory

- 1800`s two German biologists, THEODOR SCHUWANN and MATTHIAS SCHELEIDEN developed the theory that: 1. All living things are composed of cells. 2. Cells are living entities, surrounded by a MEMBRANE, that are capable of: a. GROWING, b. REPRODUCING c. RESPONDING d. METABOLIZING

Kingdoms

- 5 kingdoms: 1. Plantae 2. Animalia 3. plus Fungi 4. Protista 5. Prokaryotae - and VIRUSES. SCIENTISTS HAVE PROPOSED OTHER TAXONOMIC SCHEMES THAT HAVE FROM 5 TO MORE THAN 50 KINGDOMS.

Motility

- A cell's motility may enable it to: *Flea from a harmful environment * Move toward a favorable environment, SUCH AS ONE WHERE FOOD OR LIGHT IS AVAILABLE. - The most notable structures responsible for such bacterial movement are flagella.

Flagella

- Are extensions of a cell but are FEWER, LONGER and more WHIPLIKE than cilia - long structures that extend beyond the surface of a cell and its glycocalyx and propel the cell through its environment. - NOT ALL BACTERIA HAVE FLAGELLA - for the bacterias that do have flagella, the composition is very similar in: 1. STRUCTURE a. Filament - a long hollow shaft, about 20nm in diameter which extends out of the cell into its environment. * No membrane covers a filament. * A bacterial flagellum is composed of many identical globular molecules of a protein called FLAGELLIN. * A Flagellum lengthens by growing at its tip as the cell secretes molecules of flagellin trought the hollow core of the flagellum, to be deposited in a clockwise helix at the tip of the filament. * Bacterial flagella react to external wetness, inhibiting their own growth in dry habitats. b. Hook - At its base, a filament inserts into a curved structure, the Hook, which is composed of a different protein. c. Basal Body - Is composed of still different proteins, anchors the filament and hook to the cell wall and cytoplasmic membrane by means of a rod and a series of either two or four rings of proteins. * Together, the hook, the rod and the rings allow the filament to rotate 360 degrees. ** Differences in the proteins associated with the bacterial flagella vary enough to allow classification of species into strains called SEROVARS 2. ARRANGEMENT: a. Peritrichous - Flagella that cover the surface of the cell b. Single Polar Flagella - Are only at the Ends * other bacteria have tufts of polar flagella. c. Tuft of Polar Flagella d. Endoflagella - Spiral-shaped bacteria, called spirochetes - Have flagella at both ends that spiral tightly around the cell instead of protruding into the surrounding medium. - Form Axial Filament that wraps around the cell between its cytoplasmic membrane and an outer membrane. -Rotation of the endoflagella evidently causes the axial filament to rotate around the cell, causing the spirochee to corkscrew throught its medium. * AXIAL FILAMENT is made of ENDOFLAGELA. 3. Function -Bacterial flagella ROTATE 360 degrees. - The flow of ions (electrically charged atoms) throught the cytoplasmic membrane near the basal body powers the rotation, propelling the bacterium throught the environment at about 60 cell lengths per second - equivalent to a car traveling at 670 miles per hour - Bacteria move with a series of RUNS interrupted by TUMBLES. * RUN: COUNTERCLOCKWISE flagellar rotation produces movements of a cell in a single direction for some time * TUMBLE: Runs are interrupted by brief, abrupt, random changes in direction called Thumbles. Thumbles result from clockwise flagella, which then adjust their speed and direction of rotation. * TAXIS - Movement in response to a stimulus (Run towards to a more favorable environment) ** If more than one flagellum is present, the flagella align and rotate together as a bundle. * CHEMOTAXIS - Movement of motile cell or organism, or part of one, in a direction corresponding to a gradient of increasing or decreasing concentration of a particular substance. Movement towards food.

Prokaryotic cell

- BEFORE NUCLEUS - The distinctive feature is that they can read their DNA genetic code and SIMULTANEOUSLY MAKE PROTEINS. - Does not have a MEMBRANE surrounding its genetic material, that is DOES NOT HAVE A NUCLEUS. - Smaller than Eukaryotes - LESS complex than Eukaryotes

anaerobic respiration

- Bacterial cells that live without Oxygen - These cells can still undergo cellular respiration. The only difference is that O2 is not going to be their terminal electron ACCEPTOR. * In anaerobic respiration the same steps occur: 1. Glycolysis 2. Aceto CoA formation 3. Kreb's Cycle 4. Electron Transportation Chain - the difference here is that cytochrome doesn't add electrons to oxygen. Instead, there is a different enzyme and it is going to donates the electrons to a non- O2 (non-oxygen) source. - For many other anaerobic bacteria, they will take: • Nitrate (NO3) and reduce it to nitrite (NO2) NO3 - + 2e- + 2H+NO2 - + H2O * The NO3 is powering the ETC, turning into NO2.

Endospores (if applicable)

i.

Define morphology

shape

Why is glycolysis considered a universal metabolic pathway? What is the Entner-Doudoroff pathway? Which organisms use it? How can we use this to identify them?

- Because everybody uses glycolysis except some bacteria that undergo a different pathway, the Entner-Doudoroff Pathway. - It is a different metabolic pathway used by some bacteria: Ex.: a. PSEUDOMONAS SP (see in lab) b. VIBRIO CHOLERA (causes cholera) 3. ENTEROCOCCUS FAECALIS (see in lab) * These bacteria can be tested by testing for the presence of KDPG because they are unique for bacterias that undergo the Entner-Doudoroff Pathway. - It uses different type of glycolysis because it used different enzymes. 1. It can be identified by the enzyme: KDPG that stands for 2-keto-3-deoxy-6-phosphogluconic acid. 2. Also, glucose is broken into 2 ethanol, 2 CO2 and 1 Net ATP. - It yields LESS ENERGY than glycolysis

Cell membrane

d.

Nucleoid region

e.

Recall the various diseases that S. aures and S. pyogenes can cause.

eX. 1 S. PYOGENES - Scarlet fever is caused by an exotoxin released by Streptococcus pyogenes - Gram positive, beta hemolytic, streptococci. - Traditionally, it causes strep throat. - If that particular strep has been lysogenized, it can be carrying an erythrogenic toxin (scarlet fever toxin) which will also target the Red Blood cells. - Strains that release exotoxins can cause Scarlet fever which is characterized by a generalized rash and "strawberry tongue" - The toxin is released through the bloodstream and targets the Red blood Cells. NEUROTOXIN: AB TOXIN - Botulinum Toxin The AB toxins are toxic proteins secreted by living bacterial cells, such as the potent endospore former Clostridium botulinum. AB toxins are classified as Neurotoxins, for they interfere with the nervous system functioning. They are composed of proteins A and B that are bonded together. Protein A is the Active toxin (or the "light chain"), and the protein B is the Binding site (or "heavy chain"). Once excreted by the bacterial cell, the protein B binds to the extracellular receptors of the neural cell. The host cell then engulfs the toxin, transporting it inside by endocytosis, which creates a vesicle inside of the host cell. Components A and B then separate, and the protein A is released inside the host's neuro cell while the B component gets trapped inside the vesicle. The toxin has very specific targets inside the cell, and therefore it targets and destroy the Snap-Snare complex. As a result, the neurotransmitter Acetylcholine is not able to be released across the Synaptic Complex in order to reach the muscles, which therefore prevents muscular contractions, causing Flaccid Paralysis. Botulinum toxins are very small and they can be transported all over the body through the bloodstream, which can lead to general paralysis and even death for respiratory complication due to diaphragm failure. It is mostly transmitted by infection of the umbilical cord in neonates. It can also be caused by ingestion the toxin present in honey or in contaminated canned food (for it gives the anaerobic environment necessary for the bacteria to strive and to make its toxins). Nowadays, botulinum toxins can also be used for cosmetic purposes, such as the facial Botox that relaxes the facial muscles. Ex.2 : S. aureus - It is part of some people's normal flora - causes multiple diseases based on the portal of entry it invades • Skin: boils and carbuncles • Bone: osteomyelitis • Ingestion: Staph food poisoning • Eye: conjunctivitis - S. aureus has a leucocidin called Panton-Valentine leukocidin and it destroys white blood cells found in the bloodstream. - Inhibit BLOOD CELLS - S. aureus uses a fibrinolysin called Staphylokinase to break down the fibrin networks (which allows the tissue to remain tightly packed together) that try to localize the tissue so it can spread faster - flesh eating disease - S. aureus capsule has coagulase on it so blood clots around the cells making them "invisible" to the immune cells in the body

Cytoplasm

f.

Ribosomes

g.

Inclusion bodies

h.

Describe the composition of peptidoglycan

i.

Bacteria that forms endospores and the diseases they cause

j.

How can pH affect the growth of an organism?

• many of the metabolic reactions require enzymes in order to occur, and enzymes are proteins. Anything that affect the proteins will affect the metabolism. Therefore, in addition to temperature we need to look at pH requirements. • Most bacteria grow best in a pH range 6.5-7.5 (avg body pH is 7.4) • Pathogenic bacteria that infect humans grow best at our physiological pH (7.4) • Acids are used in foods to prevent spoilage for this reason (lemon juice, ascorbic acid- Vit. C) to prevent food from spoiling.

Phototroph

- Cyanobacteria, purple bacteria - Use energy from light (sun) for biosynthetic reactions (photosynthesis)

Recall the function of the 3 proteins in the lac operon

- Genes carried either on the genome or on the plasmid, the genes themselves are considered: -> are sequences of nucleotides (A,T,C or G's) that have a biologic function * Genes are not always proteins - Does not have always to encode for Protein production - Genes just have to give us some type of information Three kinds of genes: 1. Cistron genes: - coding genes - Gives you PROTEIN Product - Gene will be transcribed into RNA and then translated into protein. 2. Ribosomal ribonucleic acid Genes (rRNA genes): - Gives the instructions to make RIBOSOMES. 3. Promoter genes: recognition and binding sites for other molecules (promoters and operators) - Are the starting site for transcription - It does not give instruction for a protein product, but it contains information encoded in them - that is why they are considered genes. - They are recognition and binding site for RNA Polymerase and for Regulatory Proteins. 4. Operator genes: recognition and binding sites for other molecules (promoters and operators) - It does not give instruction for a protein product, but it contains information encoded in them - that is why they are considered genes. - They are recognition and binding site for RNA Polymerase and for Regulatory Proteins. Gene Regulation • Gene regulation in prokaryotic cell is very tightly regulated • Regulation helps prokaryotic cells conserve energy and resources by only producing proteins/products that it needs under specific conditions * Some of the systems of gene regulation are going to occur when environmental conditions are shifting, and as the environment shifts, the bacteria is going to shift the type of genes that they are expressing. *Bacterial cells have the ability to regulate enzymes and any other type of protein at various times during the expression of the protein. Sometimes, it is Post translational regulations: - POST TRANSLATIONAL REGULATION (after the protein is made): Bacterial cells have the ability to regulate the amount or the function of enzymes that are produced AFTER it is made. • Regulation of enzyme activity and synthesis can control the efficiency with which a cell undergoes metabolism - and metabolizes carbohydrates, lipids or any other type of source. * A cell regulates it's metabolism by regulating the enzymes that are produced - Modes of Regulation: 1. Regulation at the level of TRANSCRIPTION - Blocks formation of mRNA - If you don't have mRNA, you wouldn't get translation 2. Regulation at the level of TRANSLATION - Would block Ribosome. - mRNA would be produced, but the ribosome would be inhibited, so we would stop translation 3. Degradation of PROTEIN: - Block the protein itself. - Less efficient because at this point you would had already made a messenger RNA, and it would have already been translated into the protein and at that point the cell would go and degrade the protein so it would no longer function. - Two different types of gene regulation in a prokaryotic cell: 1. INDUCIBLE SYSTEM • Ex.: Lac Operon 1. Inducible System - We would be turning gene expression on - In an inducible system, the genes are usually not expressed, but some environmental change triggers the expression of the gene. Ex.: LAC Operon (to help the cell metabolize lactose if it becomes the only carbohydrate that is available in the environment). --> Graph: Cell number increases so total protein increases as well because the more cells you have, the more relative protein you would have. The blue line represents the specific protein Beta-Galactosidase and it is part of the LAC OPERON. It's expression is relatively low even though the number of cells increases, which indicates that the cell is not expressing that protein, until the gene gets induced (or turned on). We can turn the Lac Operon on by adding LACTOSE. By adding lactose we can see that it triggers the expression of Beta-Galactosidase. So as the number of cells increases, so does the number of Beta-galactosidase will increase as well *** The regulation of these types of systems is regulated by an operon a. OPERON - Is a way of prokaryotic cells to regulate their gene expression. Their function: • An operon is a system of gene regulation in prokaryotes (there is some evidence they are in use in lower order animals like C. elegans) • Operons have 3 major parts: 1. Promoter (upstream or before the Operator) - RNA Polymerase binds to Promoter to start Transcription. - The genes that control the transcription of the structural genes into actual proteins 2. Operator (either an inducer or a repressor) - Downstream or after the promoter - Promoter and Operator control transcription. - Regulator Protein binds to the Promoter in order to inhibit the RNA Polymerase to bind to the Promoter and to Transcribe the Structural Genes (because this is an INDUCIBLE SYSTEM, therefore, the gene expression is naturally turned off). * A Regulator Protein is NOT part of the Lac Operon, as it is made on a different part of the genome, but it is required so it can bind to the Operator and prevents the transcription of the downstream Structural Genes into protein products. - If we remove the protein, the RNA Polymerase would bind to the Promoter and go ahead and transcribe the Structural Genes. ---> Following the Transcription, you would get a piece of mRNA. The Messenger RNA can then be translated into Proteins that are coded by the structural genes. - The genes that control the transcription of the structural genes into actual proteins 3. Structural genes (downstream or after the operator) - Gives us the protein product of the operon/gives us the actual proteins PROMOTER --> OPERATOR --> STRUCTURAL GENES The first and most studied operon is the LActose operon in E. coli. B. LAC OPERON • The first (and probably most studied) operon is the Lactose (Lac) operon and it is found in E. coli (but also found in other cells) • It is an inducible system: It will be turned on when the lactose is present, but normally it is turned off • This operon regulates lactose metabolism in the absence of glucose. • Glucose is the starting product for glycolysis. • Lactose is a disaccharide, so it is necessary to break the glycosidic linkage that holds it together to release one of the glucose that is part of the lactose. • With glucose present, this operon is repressed (or turned off) - because the cell will start using the glucose that is readily available • When glucose is not present but lactose is, the operon is activated (or induced): The cell will start to express the genes that are required for lactose metabolism. • The Lac operon contains 3 structural genes: 1. The enzyme β- galactosidase • It is an enzyme that breaks the glycosidic linkage that holds the two monosaccharides together in the fructose, releasing the monosaccharide glucose • Absolutely required for Lactose Metabolism. 2. The protein lactose PERMEASE • which creates a channel in the membrane (transport protein) that allows lactose to diffuse into the cell. * If you are going to switch to lactose metabolism, you better make sure that you can get lactose into the cell - and Lactose Permease will allow it to occur • Absolutely required for Lactose Metabolism. 3. The enzyme β-galactosidase transacetylase - Just part of the Lac Operon. - If you mutate it, you can still metabolize lactose, but doesn't have a known job. * Not necessary for Lactose Metabolism. - Lac operon is inducible: only active when lactose is present and glucose is absent 1st - GLUCOSE PRESENT, LACTOSE ABSENT: - Allosteric regulator protein, which means that Regulatory Protein is normally bound to the Operator. Therefore we do not see transcription downstream when glucose is present - bc even though RNA Polymerase might bind to the promoter, the Repressor/Regulatory Protein is bound to the Operator. • Without lactose the regulator is bound to the operator • This stops mRNA from being formed 2nd GLUCOSE PRESENT, LACTOSE PRESENT ** Regulation in the level of the Lactose - When lactose is present with glucose, repression is lifted but enzyme need is limited • When glucose is absent but lactose is present, then repression is lifted and enzyme need is greater ----> What role does lactose and the regulatory protein play in regulation of gene expression? - Lactose is responsible for preventing the regulator protein from binding to the Operator. Once the regulator is released, the operator is exposed. * Lactose binds to the Repressor Protein. When it is bound to the repressor protein, the repressor protein does not bind to the operator and therefore does not inhibit the RNA Polymerase to bind to the Promoter, so it goes ahead and transcribes the Structural Genes, and, therefore protein IS made. 3rd - GLUCOSE ABSENT, LACTOSE PRESENT ** Regulation in the level of the absence of Glucose. - By removing Glucose we can remove all of the repression that is required in order to get a higher number mRNA being made. - We need Glucose to be completely absent (to clear the way for the RNA Polymerase - When Glucose is completely absent, cAMP binds to CAP, and then both of them together bind to the promoter, in order to allow the RNA Polymerase to bind more efficiently to the operon. * The Regulator Protein would be missing in this case because the lactose bound to it, inhibiting it from binding to the operon. * When the levels of glucose start to fall, the levels of cAMP start to climb and bind to CAP. **If Glucose is high, then cAMP levels is low, so it does not bind to the CAP, so then they don't bind to the promoter, and as a result RNA Polymerase cannot efficiently bind to the Promoter, so then we get very little translation even though lactose is present So... • Once the regulator is released, the operator is exposed • RNA polymerase binds and transcribes the structural genes • So then you get messenger RNA. The mRNA transcript is polycistronic- because all 3 of the structural genes are transcribed and will be made in a continuous piece of mRNA (B-Galactosidase, Permease and Transacidolase) • After the mRNA is made, each gene will be translated individually and that will give us the individual proteins • When lactose is used up, the regulator will not be repressed and it will be bound to the operator again and expression will be turned off. lac operon C. - A major advantage is that it leads to the synthesis of groups of functionally related enzymes, usually from a single mRNA transcript. Since a functional pathway must be activated in terms of all of its components, the operon affords an all-or-none response that serves efficiency. The control mechanisms existing for turning on or turning off the operon make the operon sensitive to those changes in the environment to which the bacterial cell must adjust. The ability of the cell to fashion repressors that are functional as well as repressors that are nonfunctional permits a broad variety of environmental triggers to exert their effects. Inducible systems may be activated by agents that tie up the repressor, and constitutive systems may be shut off by corepressors that activate initially ''blank'' repressors. Further fine tunning is achieved by the participation of CAP protein and cyclic AMP in the activation of the promoter site. Moni: - The operon system would be an advantage to bacterial cells because it leads to the synthesis of groups of functionally related enzymes from a single mRNA transcript and also aids cells to adjust to the production level of certain enzymes. This means that cells are able to regulate the expression of genes encoding the enzymes. - The operon is of advantage to the bacterial cell in that it acts as a balance in the process of gene expression. An operon itself is a unit of linked up genes that regulate other genes responsible for protein synthesis. That being said, the operon is known to aid in the regulation of gene expression. -What role does lactose and the regulatory protein play in regulation of gene expression? - Lactose is responsible for preventing the regulator protein from binding to the Operator. * Lactose binds to the Repressor Protein. When it is bond to the repressor protein, the repressor protein does not bind to the operator and therefore does not inhibit the RNA Polymerase to bind to the Promoter and transcribe the Structural Genes, and, therefore protein IS made. cAMP - • Cyclic AMP (cAMP) is a cell signaling protein CAP - • Catabolite activator protein (CAP) 3. REPRESSION SYSTEM • Ex.: Quorum Sensing • Repression systems are always "on", unless they are turned off by an specific product * Almost all of the amino acid operons (the enzymes that allow the cells to make different amino acids) those are almost always Repression Systems * The bacterial cell will continue to make amino acids untill the concentration of the amino acids is hight. • The arginine operon synthesizes the amino acid arginine • When free arginine levels are too high and has reached its maximum (meaning the cell is not using the arginine anymore) it can bind to the repressor and therefore the Repressor gets activated, and binds to the Operator in order to turn OFF the production of amino acid so that amino acid will no longer be made (The repressor then binds the operator and stops transcription) - As the concentration of amino acids begin to fall, the operon will be turned back on and the amino acid will be made again --> This is a repression system: it is normally on, it is normally being expressed, and we are normally making the amino acid, but if the levels get to hight, the cell will turn this operon off --> Only the levels of Arginine are responsible for regulating the amount of expression that we see. What is quorum sensing? - Another way that prokaryotic cells can regulate the types of genes that they are expressing. * Bacteria that is able to undergo quorum sensing have genes that are regulated by population density (The more cells you have, the more likely these genes are to be turned on) • Some prokaryotes have regulatory pathways that are only triggered when the population reaches a certain density • The quorum sensing genes are controled by something known by "Autoinducer" * cells are always producing autoinducer in very low concentration. • Autoinducer diffuses out of the cell and freely diffuse into neighboring cells at very low levels. * Concentration is population dependent: if more cells= you have more autoinducer • At some point, if you have enough cells in the same giving area, the concentration of autoinducer will be high enough, and that will allow the Autoinducer to bind to the Activator Protein. * The Activator Protein allows us to trigger the transcription of quorum specific genes --> Population triggering gene expression: when you have enough cells together in a give area, they start to express completely different genes. * It is special and unique to certain types of bacteria. ** The danger about quorum sensing is that a lot of times the activator proteins trigger the transcription of VIRULANCE GENES. (The more bacteria you have the more virulent they become) Ex.: P. aeruginosa uses 4-hydroxyl alkyl quinolines as an autoinducer • Reaching quorum triggers production of genes that allow it to go from planktonic to biofilm- increases pathogenicity and inhibits antibiotic penetration Ex.2: A. fischeri uses homoserine lactone as an autoinducer - Turns on luxbioluminescence gene - Lux operon (Lux = light): ex. bobsquid with the bacteria that glow in the dark and allow he squid to run from predators. What is quorum sensing?

Explain what an enzyme is.

- Many biological reactions require the use of an enzyme. - Enzymes are biological catalysts that lower the activation energy of a reaction. It means we would need to put in less energy into the system in order to get the reaction to move forward - Are made of organic materials. They are carbon containing compounds that in most living things are PROTEINS - Enzymes only affect the rate of the reaction. It doesn't affect anything else. - Enzymes can be reused over and over again to allow the reactant to make product. - When an enzyme is bound to the reactants, we call it the enzyme substrate complex because the substrate is essentially a REACTANT. - It contains: 1. APOENZIME - The protein- portion - enzymes without their cofactors 2. CO-FACTOR - a nonprotein chemical compound that activates and assists enzymatic reactions. - Can be: a. Inorganic Co-factor * Ion (Metal ion): Mg+, Fe, Ag, Au * Can be found inside of the protein part of the enzyme b. Co-enzymes * Organic in nature. * They contain at least one carbon and a lot of times the co-enzymes found in the apoenzymes (protein part of the enzyme) are vitamins, such as vitamin A, D, C. 3. HOLOENZYME - Apoenzyme + Co-enzyme = Holoenzyme - When the apoenzyme and the co-enzyme are joined together we have a holoenzyme. - Holoenzyme is an active enzyme that is ready to accept a substrate. - If the temperature is optimal and if the pH is optimal and if there is free enzyme available for the substrate to bind, then the enzyme can catalyze a reaction - In a chemical reaction controlled by an enzyme, one or more substrate molecules fit into the catalytically active amino acids in the active site (where you would find the cofactor as well) of the enzyme to form an enzyme-substrate complex. The substrate molecules are held in such a way that a reaction takes place. Product molecules are produced and released while the enzyme is unchanged. - Ex.: When a peptidoglycan gets into the active site of an enzyme (where the apoenzyme and the cofactor can be found), the enzyme-substrate complex break the bond of the disacharide, creating a EXORGONIC reaction, which releases energy. --> Enzyme activity can be affected by: 1. TEMPERATURE - Enzymes have protein portions. - both temperature and pH have the ability to denature the protein. * if it is cold, the enzymes won't denature. What will happen is very slow molecules. Therefore the rate in which the reactions occur is very low because the substrate is not going to collide very often with the enzyme and catalyze the reaction. * 37/body temperature is the peak point. * if it is too hot, there is a decline in activity. The hydrogen bonds begin to break and the protein unfolds and the three dimensional shape is lost and the protein is denatured (40 degrees + more or less) 2. pH - Enzymes have protein portions. - both temperature and pH have the ability to denature the protein. - pH * A shift of pH will affect enzyme activity * If the environment becomes too basic or too acidic, the protein will be denatured and the enzymes won't be able to function or catalyze its reaction * 7< = Acid * 7= Neutral * 7> = Basic 3. Saturation - Substrate concentration has a saturation point. - It means that the enzyme activity is not increasing anymore, that is, we have filled every single enzyme in the system with substrate (saturated). So the reaction we have cannot go any faster because all of the enzymes we have are already being used. - Cells can control the rate of reaction by controlling the enzyme concentration.

Identifying Microorganisms

- Scientists usually use: 1. PHYSICAL CHARACTERISTICS a. Morphology b. Physical Appearance 2. BIOCHEMICAL TESTS - Can only be used to identify microbes grown under laboratory conditions. - Biochemical tests include procedures that determine an organism's ability to: a. FERMENT various CARBOHYDRATES b. utilize varions SUBSTRATES, such as specific AMINO ACIDS, STARCH, CITRATE, and GELATIN c. Produce WASTE PRODUCTS, such as HYDROGEN SUFIDE (H2S) - Many tests require that the microorganisms be CULTURED (grown) for 12-24 hours, though this time cab be greatly reduced by the use of rapid identification tools. 3. SEROLOGICAL TESTS - Serology is the study of SERUM - SERUM is the liquid portion of blood after the clotting factors have been removed and an important site of antibodies. -In its most practical application, serology is the STUDY OF ANTIGEN-ANTIBODY REACTIONS in laboratory settings. * ANTIBODIES are immune system proteins that bind very specifically to TARGET ANTIGENS. - Many microorganisms are ANTIGENIC, that is, within a HOST ORGANISMS they TRIGGER an IMMUNE RESPONSE that results in the PRODUCTION ANTIBODIES. * EX. suppose that a scientist injects a sample of BORRELIA BURGDORFERI, the bacteria that causes Lyme disease, into a rabbit. The bacterium has many surface proteins and carbohydrates that are antigenic because they are foreign to the rabbit. The rabbit responds to these foreign aitgens by producing antibodies against them. * These antibodies can be ISOLATED from the rabbit's serum and concentrated into a solution known as ANTISERUM. - ANTISERA bind to the antigens that triggered their production. - In a procedure called an AGGLUTINATION TEST, antiserum is mixe with a sample that potentially contains its target cells. If the antigenic cells are present, antibodies in the antiserum will clump (AGGLUTINATE) the antigen. * Other antigens, and therefore other organisms, remain unaffected because antibodies are highly specific for their targets. -Antisera can be used to distinguish among species and even among strains of the same species. 4. PHAGE TYPING - BACTERIOPHAGES (or simply PHAGES) are VIRUSES that infect usually destroy BACTERIAL cells. * Just as antibodies are specific for their target antigens, phages are SPECIFIC for the HOSTS they can INFECT. - PHAGE TYPING, like serological testing, works because of such specificity. One bacterial strain may be susceptible to a particular phage (virus) while a related strain is not. - In Phage typing, a technician spreads a solution containing the bacterium to be identified across a solid surface of growth medium and then adds small drops of solutions containing different types of bacteriophage (viruses). * Wherever a specific phage (virus) is able to infect and kill bacteria, the resulting lack of bacterial growth produces within the bacterial lawn a clear area called PLAQUE. --> A MICROBIOLOGIST CAN IDENTIFY AN UNKNOWN BACTERIUM BY COMPARING THE PHAGES THAT FORM PLAQUES WITH KNOWN PHAGE-BACTERIA INTERACTIONS. 5. ANALYSIS of NUCLEIC ACIDS - The sequence of nucleotides in nucleic acid molecules (either (DNA or RNA) provides a powerful tool for CLASSIFYING and IDENTIFYING MICROBES. -In many cases, nucleic acid analysis has confirmed classical taxonomic hierarchies. * with techniques of Nucleotide Sequencing and comparison such as POLYMERASE CHAIN REACTION (PCR). Determining the percentage of a cell's DNA that is guanine and cytosine, a quantity refered to as the cell's G + C content (or G+C Percentage), has also become a part of prokaryotic taxonomy. Scientists express the content as follows: G+C/A+T+G+C x 100 G+C content varies from 20% to 80% among prokaryotes. Often (but not always) organisms that share characteristics have similar G+C content. Organisms that were once thought to be closely related but have different G+C percentages are invariably not as closely related as thought.

Wavelength

- the distance between two corresponding parts of a wave.

infection.

- the entry establishment and growth (multiplication) of pathogenic microorganisms within a host. Occur in 3 Steps: 1. Contact a. Endogenous, or b. Exogenous. 2. Invasion 3. Infection - followed by either: a. Asymptomatic Infection (where the Carrier State develops), or b. Immunity/Repair of damage, or c. Morbidity/Mortality occur- the entry establishment and growth (multiplication) of pathogenic microorganisms within a host. Occur in 3 Steps: 1. Contact a. Endogenous, or b. Exogenous. 2. Invasion -> ATTACHMENT - Third in the steps of the ability to cause disease - The organism has to attach - it is absolutely required. • Once in, the bacteria must adhere to cause disease - Where/ to what do microorganism attach? To the extracellular matrix of the host cells. If it does not adhere and attach, the bacteria is not able to cause disease. - Name some components that are on the eukaryotic cell and provide points of attachment for the microbe. 1. Glycocalyx - binding to the extracelullar matrix of the host cell 2. Pilli 3. Infection - followed by either: a. Asymptomatic Infection (where the Carrier State develops), or b. Immunity/Repair of damage, or c. Morbidity/Mortality occur - The rate and severity of the infectious disease is going to be affected by 3 different factors: 1. Portal of entry 2. Number of organisms - Or infectious dose - or Number of Organisms - The second factor that influence the severity of the infection - It is the Average Minimum number of microbes required for infection to proceed * Microbes with small IDs have greater virulence because it requires fewer microbes to cause disease 3. Virulence of organisms - The third factor that influence the severity of the infection - It is the virulence of the organism itself. * Is one serovar more virulent than another? Ex.: Comensalistic E. Coli and Hemorrhagic E. coli. If you would have to choose to be infected with one of them, you would choose the comensalistic because it is not going to harm you. Factors • Virulence factors enhance pathogenicitiy • Pathogenicity is the ability of the microbe to cause disease while virulence factor is the degree to which an organism causes damage to the tissue. * Virulence factor that helps enhance pathogenicity because they make the bacteria more able to cause disease. • Three major classes of Virulence Factors: 1. EXTRACELLULAR ENZYMES • One of the three major classes of Virulence Factors. • Extracellular enzymes are secreted by a pathogen into the environment • Most of the extracellular enzymes secreted by the pathogen will damage the host cell tissue. • Most dissolve structural chemicals in the body and help invasion, maintenance of infection or immune evasion A. Hyaluronidase - Extracellular enzyme 1 - (spreading factor) - Enhance SPREADING and MOVEMENT - Degrades hyaluronic Acid, which is part of the extracellular matrix that holds the cells together. - Dissolve parts of the Extracellular Matrix and parts of the Basement Membrane that helps hold cells together. By doing so, it allows the bacteria to penetrate in between the cells and down into the deeper tissues. - Important to the virulence and pathogenesis of staphylococci, clostridia, streptococci, and pneumococci B. Collagenase Clostridium - Extracellular enzyme 2 - Enhance SPREADING and MOVEMENT by dissolving Collagen that is usually present in the basement membrane of the Epithelial Cells. - can use collagenases to dissolve collagen. - Dissolve parts of the Extracelullar Matrix and parts of the Basement Membrane that helps hold cells together. By doing so, it allows the bacteria to penetrate in between the cells and down into the deeper tissues. - Medically used to help treat types of hyperflexion (which happens when the joints can't be relaxed) C. Leucocidin - Extracellular enzyme 3 - Inhibit BLOOD CELLS - destroys/lyses white blood cells (WBC) and leukocytes- the cells of the immune system. - Increase virulence by inhibiting the immune response - S. aureus has a leucocidin called Panton-Valentine leukocidin and it destroys white blood cells found in the bloodstream. D. Fibrinolysin - Extracellular enzyme 4 - Inhibit BLOOD CELLS - S. aureus uses a fibrinolysin called Staphylokinase to break down the fibrin networks (which allows the tissue to remain tightly packed together) that try to localize the tissue so it can spread faster - flesh eating disease E. Kinases - Extracellular enzyme 5 - Inhibit BLOOD CELLS - digests blood clots and enhances invasion * If a bacteria can break through a blood cloth it can enhance faster - Staphylokinase and streptokinase F. Coagulase - Extracellular enzyme 6 - Inhibit BLOOD CELLS - causes clot formation in blood around the bacterial cells. - S. aureus capsule has coagulase on it so blood clots around the cells making them "invisible" to the immune cells in the body 2. TOXINS - The second type of Virulence Factor that a number of bacteria carrie. - Unlike the Extracellular Enzymes, toxins are secreted by living bacteria cells into that causes damage to the cell by harming tissues or triggering host immune responses that enhance tissue damage. • Toxins are generally named by the tissue type they target - Toxins are very small and they have very specific targets inside the cell. - They are very potent even in very small doses - toxin will be made in one localized area where the bacteria is but it will spread throughout the body through the bloodstream - There are three types: 1. CYTOTOXIN a. Hemolysin - 1st kind of toxin. - Hemolysin is what lyses and destroys red blood cells - These toxins are what gives rise to the hemolysis patterns that we see in the blood agar plates. - Staphylococcus sp. have hemolysin which allows them to display beta-hemolysis patterns on blood agar plates - Alpha-hemolysin is a protein monomer. - The monomer subunit bind to the outer membrane of RBCs to form a pore. - Monomers oligomerize to form a water-filled transmembrane channel (water will rush in through the pore inserted in the Red Blood Cell membrane causing osmotic swelling) - It causes an irreversible osmotic swelling, which is responsible for the cell lysis b. Erythrogenic - Scarlet fever is caused by an exotoxin released by Streptococcus pyogenes - Gram positive, beta hemolytic, streptococci. - Traditionally, it causes strep throat. - If that particular strep has been lysogenized, it can be carrying an erythrogenic toxin (scarlet fever toxin) which will also target the Red Blood cells. - Strains that release exotoxins can cause Scarlet fever which is characterized by a generalized rash and "strawberry tongue" - The toxin is released through the bloodstream and targets the Red blood Cells. 2. NEUROTOXIN - AB Toxins a. Clostridium Botulinum - prevents contraction by preventing the release of Ach from the nerve into the muscle. The AB toxins are toxic proteins secreted by living bacterial cells, such as the potent endospore former Clostridium botulinum. AB toxins are classified as Neurotoxins, for they interfere with the nervous system functioning. They are composed of proteins A and B that are bonded together. Protein A is the Active toxin (or the "light chain"), and the protein B is the Binding site (or "heavy chain"). Once excreted by the bacterial cell, the protein B binds to the extracellular receptors of the neural cell. The host cell then engulfs the toxin, transporting it inside by endocytosis, which creates a vesicle inside of the host cell. Components A and B then separate, and the protein A is released inside the host's neuro cell while the B component gets trapped inside the vesicle. The toxin has very specific targets inside the cell, and therefore it targets and destroy the Snap-Snare complex. As a result, the neurotransmitter Acetylcholine is not able to be released across the Synaptic Complex in order to reach the muscles, which therefore prevents muscular contractions, causing Flaccid Paralysis. Botulinum toxins are very small and they can be transported all over the body through the bloodstream, which can lead to general paralysis and even death for respiratory complication due to diaphragm failure. It is mostly transmitted by infection of the umbilical cord in neonates. It can also be caused by ingestion the toxin present in honey or in contaminated canned food (for it gives the anaerobic environment necessary for the bacteria to strive and to make its toxins). Nowadays, botulinum toxins can also be used for cosmetic purposes, such as the facial Botox that relaxes the facial muscles. b. Tetanospasmin - Causes permanent contraction of the muscles by preventing Ach to leave the muscle. • Clostridium tetani produces the toxin called tetanospasmin • Endospore former and Anaerobic • An A-B toxin • Causes tetanus (Lockjaw) • Neurotoxin causes uncontrolled muscle contraction, especially in large muscle groups. * It prevents the Ach to leave the muscle • clenching of jaw, extreme arching of the back, respiratory paralysis leads to death for the diaphragm remains contracted. • C. tetani is a common inhabitant of soil and in the intestinal tract of animals (esp. horses). • Endospores are ubiquitous (which means "found everywhere"). • Spores enter puncture wounds, burns, umbilical stump, frostbite, necrotic tissue, anaerobic conditions • Not a communicable disease because it is not transmitted from one person to the other - the person must get endospores into the wounds each time, in order to develop the illness. • Treatment with antitoxin therapy- IgG against tetanus toxin (targets the toxin, not the bacteria) * Vaccine works against the toxins - it tries to prevent the toxin to reach the nervous system and targets the toxin before it causes the uncontrollable reactions. * It is very necessary to remove the toxin from the bloodstream first hand, in order to remove the symptoms it causes (and help the diaphragm to work properly, for example). The bacterial infection is secondary and it is easily treated (killed) with antibiotics. • Can control the infection with antibiotics (penicillin, tetracycline) treat spasms with muscle relaxers, and ease respiratory distress with ventilator • Prevented with vaccination with tetanus toxoid- toxin is inactivated by can still produce immune response protection last 10 years- are you up to date? Neonatal tetanus acquired though infection of umbilical stump 3. ENTEROTOXIN a. Staphylococcal enterotoxin B • Targets cells in the GI Tract • The things that cause all the tissue damage that give rise to the symptoms that we see during food poisoning • Staphylococcal enterotoxin B • Bacteria will cause infection when ingested (portal: mouth) • Causes rapid onset food poisoning (30 min to 2 hours) * Onset is so fast because staph will produce the toxin and the toxin will be in the food and if you eat a large amount of the food, you will get a dose large enough to get sick. ** We don't have to wait for the bacteria to get into your GI tract and replicate because you would already be injesting the toxin that was already made. • Severe diarrhea, nausea and intestinal cramping often starting within a few hours of ingestion • Toxin is heat stable - even if you heat the food up, the toxin will persist and still be in the food even after the bacteria have been killed * Normally happens in buffets because the food is allowed to stay sitting out, which allows the staph to produce the toxin • Gastroenteritis (inflammation of the TI tract) results from the release of our own cytokines, as a result of the toxin, that causes the inflammation that distroies the tissues B. LIPOPOLYSACHARIDE (LPS) - Generally, only 1 type: referred to as Lipopolysaccharide (LPS) found in the outer membrane of the Gram- neg bacterial cell wall • The Lipid A portion of the endotoxin that is the most damaging. • Endotoxin is released every time a bacterial cell lyse and the LPS is released into the infection site or bloodstream * If you use antibiotic and kill off a large number of GRAM-NEGATIVE bacteria, the LPS can be released into the bloodstream and it can trigger a number of very severe reactions, such as: • Causes systemic effects such as fever, inflammation, diarrhea (if in the GI tract), hemorrhage or endotoxic shock which is fatal • LPS Endotoxin will also released every time a cell divides, although in very small amount. • It will also be released every time a gram-negative cell is phagocytized by our own immune cells (macrophages) * They are EXOTOXINS because they were released from a LIVING CELL. - toxins secreted by LIVING bacterial cells into infected tissues • Exotoxins are very small proteins • Very specific for target cell and extremely powerful even in the smallest doses • Toxins are generally named by the tissue type they target Ex.: Cytotoxins, Neurotoxins and Enterotoxins. 3. ANTIPHAGOCYTIC FACTORS • The last VIRULENCE FACTOR • Antiphagocytic factors are Virulence Factors because they help enhance immune evasion * If a cell has an antiphagocytic factor, it is able to avoid being engulfed and removed by the immune system. • Macrophages are one of the first lines of defense and response when removing any type of microorganism (INNATE IMMUNE RESPONSE) • They phagocytize (form of endocytosis) foreign cells, destryoing them and removing them from the body. • S. pneumoniae, S. typhi, N. meningitidis, and C. neoformans have a very large glycocalyx (either capsule or slime) that protect them, which prevents them from being phagocytized - and if they can't be phagocytized, they can't be removed, so they will remain in the tissue and cause an infection. • Gonorrhea also has an antiphagocytic factor: It can produce chemicals that inhibit the fusion of lysosomes to the phagocytic vesicle * While gonorrhea can be engulphed by the macrophages, the macrophages can't break them down with the lysosomes, so gonorrhea will pass through the macrophages intact. - There are four distinct stages of clinical infections (morbidity): 1. INCUBATION PERIOD - Asymptomatic - or very few symptoms (if any) 2. PRODROMAL STAGE - Replication begins - Tissue damage begins - Nonspecific complains phase ("I don't feel well", "I feel ache" - there is nothing specific, but the patient doesn't feel well and doesn't feel like themselves) 3. PERIOD OF INVASION - when replication is at its hight. - When most of the tissue damage occurs - When the disease-specific symptoms and signs start to appear * this is when diagnostics start to be possible to be done 4. CONVALESCENT PERIOD * If patience survives - When replication stops - Tissue repair begins - Symptoms start to vanish - Diseases are also classified according to their severity. 1. Acute Infection - an infection that develops rapidly, but only lasts a short time • Flu, common cold, strep throat, sinusitis 2. Subclinical (asymptomatic) - or Subclinical - the infection does not cause any apparent symptoms, but the person may be a carrier * The carrier can pass the disease without knowing it because they don't have any signs of the disease. 3. Latent - causative agent (the microorganism) remains inactive for a period of time before reactivating and causing disease • Shingles, syphilis, some forms of TB 4. Chronic Infection - an infection that develops slowly and persists over a longer period of time * the bacteria or the virus (the causative agent) is always there. You can get better, but you will never be able to clear it completely out of your body. • Mononucleosis (Kissing Disease), HSV, HIV, Hepatitis B, TB

Dark-Field Microscope

- The specimen is made to appear light against a dark background

Phase Microscopes

- Use the alignment or misalignment of light waves to achieve the desired contrast between a living specimen and its background

Explain what transformation, conjugation and transduction are.

-> vertical gene transfer - Going from the parent cell to the daughter cell that arrizes from division vertical gene transfer ->horizontal gene transfer - another method for genetic diversity in bacteria comes as a result form horizontal gene transfer • Genetic diversity in bacteria can also come as a result of recombination • Horizontal gene transfer occurs when instead of going from parents cells to daughter cells, the DNA from one bacterium is transferred to another bacterium instead and this results in a change in the bacteria * The cell that is donating the piece of DNA is not replicating. It is only giving away a piece of its own DNA to another bacterial cell. (-> Extrachromosomal elements are located extra (outside) the chromosome. Example: PLASMID - A small piece of circular DNA that had a few functioning genes on it. They are located outside the chromosome/outside the bacterial genome.) - Three different ways to form Horizontal Gene Transfer: 1. CONJUGATION - A type of Horizontal Gene Transfer - genetic material shared between bacterial same species * Usually, but NOT always, a Plasmid. a. F+ Conjugation • Gram- and Gram+ can conjugate • Gram- use a plasmid called the F (fertility)- plasmid • if the F-plasmid is present, it allows the cell to form a pilus * Pillus is a hollow tube that allows the bacteria to pass plasmids back and forth • Plasmids can be conjugative or mobilizable: A. CONJUGATIVE PLASMID - will have everything that you need for conjugation to occur on it (at least part of it will have the instructions for the Pilis on it). * It B. MOBILIZABLE PLASMID - can be passed, so we can transfer it, but it lacks the information for the pilus. So there is no way for the cell to pass this mobilizable plasmid unless it already has the F Plasmid. --> First, the bacterial cell has to have this F+ plasmid in order for the plasmid to move. When F factor (a plasmid) is transferred from a donnor (F+) to a recipient (F-), the F- cell is converted into an F+ cell. b. HFR Conjugation • High frequency of recombination (HFR) occurs when the F plasmid is integrated into the cell's chromosome * It is no longer an extrachromosomal element. Now it is part of the cell's genome. • Cell joins to F- cell through a mating bridge • Hfr cell which contains the F gene will join to the F-, or the recipient cell throught a mating bridge (which is a different type of pilus) • Once the HFR Cell is joined to the F- recipient cell, the HFR cell will transfer a piece of its genomic DNA into the F- cell. * The cell receives only a partial (incomplete) copy of the F plasmid. • When the two cells break, the F- recipient will have a new piece of genomic DNA from the FHR cell, but because it receives only a partial copy of the F+ Plasmid, it will not be able to conjugate * The recipient gets genomic DNA * Not the ability to conjugate 2. TRANSFORMATION * acquisition of genetic material from the environment - A type of Horizontal Gene Transfer - Is acquisition of genetic material (DNA) from the environment • Pieces of DNA are acquired from the environment * DNA can get into the environment because perhaps another bacterial cell has lyzed. • Only competent cells can accept DNA from the environment * Bacterial cells can be made into competent cells by providing them with plasmids that have the correct enzymes and receptors in them. • If the cell is competent, then it will have DNA binding proteins in its surface that allows it to accept the DNA • This DNA fragment is incorporated into the bacterial chromosome (genomic) Transformation 3. TRANSDUCTION * It requires specific types of bacterial viruses (phages) to donate a gene and transmit genetic material - A type of Horizontal Gene Transfer - It requires specific types of bacterial viruses (phages) to donate a gene (infect bacterial cells) and transmit genetic material. - Can be Two types: A. GENERALIZED TRANSDUCTION or • If a specific sequence of the bacterial cell are transferred, it is called specialized transduction • Specialized transduction the genes that are usually being transferred from the bacterial cell are toxins to non-pathogenic cells * This allows non-pathogenic cells to become pathogenic B. SPECIALIZED TRANSDUCTION - Bacteriophages have two types of life cycles: 1. Lysogenic Cycle *The lysogenic cycle: The phage infects a bacterium and inserts its DNA into the bacterial chromosome, allowing the phage DNA (now called a prophage) to be copied and passed on along with the cell's own DNA. • Bacteriophages that undergo the lysogenic cycle integrate into the bacterial genome • The genomic material in the phage becomes part of the bacterial genome 2. Lytic Cycle • The phage genome can then hop back out of the host cell (lytic cycle) and take pieces of the host DNA with it (random) • We call this generalized transduction * The lytic cycle: The phage infects a bacterium, hijacks the bacterium to make lots of phages, and then kills the cell by making it explode (lyse). Transduction

Explain the composition and function of all of the parts of the prokaryotic cell

2.

iii. Describe the components that are specific to a Gram +, Gram - and acid fast cell wall

2.

Recall what an exotoxin is and specific examples cited in class.

24.

Recall the stages of phagocytosis and what happens during each.

28.

Compare and contrast synthetic and complex media

1. chemically defined media - or synthetic media - It has pure organic and inorganic compounds in an exact known formula. - Farely rare 2. complex media - or non-synthetic media - More common. - What ingredients make a medium complex? - It is not an exact chemical formula because it contains undefined extracts from plants, animals, or yeasts. * Examples are blood, serum, bovine heart infusions, meat extracts etc * we don't know every single component that makes it up and we don't know its concentration. They vary from lot to lot.

If organic chemicals can be made even in the ABSENCE of life, what is the difference between living thing and a nonliving thing?

- Biologists agree that all living things share at least 4 process of life: 1. GROWTH - Living things can grow; that is, they can increase in size 2. REPRODUCTION - Organisms normally have the ability to reproduce themselves. Reproduction means that they increase in number, producing more organisms organized like themselves. Reproduction may be accomplished asexually (alone) or sexually with gametes (sex cells) * Note that Reproduction is an increase in NUMBER, while reproduction is increase in SIZE. ** GROWTH and REPRODUCTION often occur simultaneously. 3. RESPONSIVENESS - All living things respond to their environment. They have the ability to change themselves in reaction to changing conditions around or within them. Many organisms also have the ability to move toward or away from environmental stimuli - response called TAXIS. 4. METABOLISM - Metabolism can be defined as the ability of organisms to take in nutrients from outside themselves and use the nutrients in a series of controlled chemical reactions to provide the energy and structures needed to grow, reproduce and be responsive. Metabolism is a unique process of living things; NONLIVING THINGS CANNOT METABOLIZE. Cells store metabolic energy in the chemical bonds of ADENOSINE TRIPHOSPHATE (ATP) * Organisms may not exhibit these four processes at all times.

Explain the role of temperature in growth and related descriptions (e.g. maximum temp, cardinal temp, etc)

- CARDINAL TEMPERATURES * the minimum, optimum, and maximum temperatures at which an organism grows. * Usually from ROOM TEMPERATURE to BODY TEMPERATURE. - MINIMUM GROWTH TEMPERATURE * the lowest temperature at which a given species of bacteria will grow * Listeria monocytogenes is a facultative psychrophile - MAXIMUM GROWTH TEMPERATUE * the highest temperature at which a given species of bacteria will grow - OPTIMUM TEMPERATURE * the temperature at which a given species of bacteria grows the best ----- A. PSYCROPHILES - Cardinal temperature: COOLER * organisms the thrive in cold conditions B. MESOPHILES - Cardinal temperature: MODERATE * organisms that thrive at moderate temperature * The optimal temperature of most pathogenic mesophiles is normal human body temperature * Most microorganisms that infect humans are MESOPHILES ( from room temperature to body temperature) Ex.: Streptococcus pneumoniae C. THERMOPHILES - Cardinal temperature: HOT * organisms that thrive at hot temperature * Thermophiles thrive where other organisms would denature. Ex.: Thermus aquaticus - over boiling is where it thrives D. ACIDOPHILES 1. What is it? - acid loving bacteria are tolerant to low pH 2. Where would it be found? - mostly in the stomach 3. Can a human pathogen be an acidophile? - H. Pylory - cause stomach acid ulcers Ex.: The protist Euglena mutabilis lives in enviornments where the pH is between 0.0-1.0, which is purely hydrochloric acid E. HALOMONADACEA - Live in arsenic based compounds (which is poisonous for human cells) F. HALOPHILES - are organisms that can grow at relatively high salt concentrations G. OSMOTOLERANT - organisms that can live in a wide range of solute ranges ( from very hypertonic to very hypotonic concentrations) Ex.: S. aureus grows on the selective medium manitol salt agar because it can tolerate the salt concentration - while other organisms can not - because it is highly osmotolerant

Describe what amphibolism is and what products it can be used to make.

- CROSS OVER BETWEEN ANABOLISM AND CATABOLISM - Because bacterial cells are unicellular, they have to be very efficient during their metabolic reactions. - As a result of this, they rely on something called AMPHIBOLISM. - Amphibolism is the ability to take intermediates that are formed during catabolic or anabolic reactions and use them for other types of pathways, or other types of metabolic reactions inside the cell. - Intermediates from glycolysis and Kreb's cycle can be used to synthesize amino acids. a. To go from carbohydrate to Amino Acids, there are three steps: 1. Amination - Add the the amino group 2. Transamination - meaning we can add more than one amino group (N) 3. Deamination - where the Amino group is removed if necessary b. To form Carbohydrates Polysaccharides are synthesized from an activated form of glucose (UDPG or ADPG) • UDPG is the precursor for NAM, NAG and LPS • Glucogenesis is formation of glucose for catabolism • Synthesized by reversal of glycolysis • Nucleic acids can be synthesized from glucose by decarboxylation (removal of the carboxyl group) of the glucose. So glucose can form a precursor for the nucleic acids that the cells would need to either replicate DNA or make messenger RNA for protein translation.

Acid-Fast Stain

- DIFFERENTIAL STAIN - NOT WATER-SOLUBLE (for waxy lipids) - Stains cells of the genera MYCOBACTERIUM and NOCARDIA (which causes many human diseases, including Tuberculosis, leprosy, and other lung and skin infections.) - Cells of these bacteria have large amounts of WAXY LIPID in their cell walls, so they do not readily stain with the water-soluble dyes used in Gram staining - Developed by Franz Ziehl (1857-1926) and Friedrich Neelsen (1854-1894) in 1883. Their procedure is as follows: 1. Cover the smear with a small piece of tissue paper to retain the dye during the procedure. 2. Flood the slide with the red primary stain, CARBOLFUCHSIN, for several minutes while warming it over steaming water. * In this procedure, heat is used to drive the stain through the waxy wall and into the cell, where it remains trapped. 3. Remove the tissue paper, cool the slide, and then decolorize the smear by rinsing it with a solution of HYDROCHLORIC ACID (pH < 1.0) and ALCOHOL * the bleaching action of acid-alcohol removes color from both non-acid-fast cells and the background. * Acid-fast cells retain their red color because the acid cannot penetrate the waxy wall. * The name is derived from this step; there is, the cells are colorfast in acid. 4. Counterstain with METHYLENE BLUE, which contains only bleached, non-acid-fast cells. - The Ziehl-Neelsen acid-fast staining procedure results in pink acid-fast cells, which can be differentiated from blue non-acid-fast cells, including human cells. - The presence of ACID-FAST BACILLI (AFBs) in sputum is indicative of mycobacterial infection.

Gram Stain

- DIFFERENTIAL STAIN - uses WATER-SOLUBLE DIES - Hans Christian Gram developed it in 1884. -- Differentiates between two large groups of microorganisms: PURPLE-staining GRAM POSITIVE PINK-staining GRAM NEGATIVE a. SMEAR - Should come from FRESHLY GROWN BACTERIA * Gram Staining works better with YOUNG cells because old cells bleach more easily than younger cells can therefore appear pink, which makes them appear to be Gram-negative cells b. HEAT Fix c. Gram staining - Assuming that the smear contains both purple Gram-positive and pink Gram-negative colorless bacteria, the classical Gram staining procedure has to follow four steps: 1. Flood the smear with the basic dye crystal violet for 1 minute and then rinse with water. *Crystal violet, which is called the PRIMARY stains, colors all cells. 2. Flood the smear with an iodine solution for 1 minute and then rinse with water. * Iodine is a MORDANT, a substance that binds to a dye and makes it less soluble. After this step, all cells remains PURPLE. 3. Rinse the smear with a solution of ethanol and acetone for 10 to 30 seconds and then rinse with water. * this solution which acts as a DECOLORIZING AGENT, breaks down the thin cell wall of Gram-negative cells, allowing the stain and mordant to be washed away; these Gram-negative cells are now colorless. Gram-positive cells, with their thicker cell walls, remain purple. ** 95% ethanol may be used to decolorize instead of the ethanol-acetone mixture 4. Flood the smear with SAFRANIN for 1 minute and then rinse with water. * this red COUNTERSTAIN provides a contrasting color to the primary stain. Although all types of cells may absorb safranin, the resulting pink color is masked by the darker purple dye already in Gram-positive cells. After this step, Gram-negative cells now appear PINK whereas Gram-Positive cells remain PURPLE ** In a 3 step variation, SAFRANIN dissolved in ETHANOL simultaneously decolorizes AND COUNTERSTAINS - After the final step, the slide is blotted dry in preparation for light microscopy.

Calculate the potential energy in a given Redox couple.

- FREE ENERGY - It is the energy that is available to do work. - The change in the free energy describes the kind of reaction we are looking at. 1. ENDERGONIC REACTION - A non-spontaneous chemical reaction in which free energy is absorbed from the surroundings. - Energy was put in and the final products have greater energy than the beginning of the reactions. - Energy was put INTO this system. - Has a POSITIVE DeltaG: DeltaG = +436 J/mol - Bond BUILDING (because we need energy to build a bond) 2. EXERGONIC REACTION - A chemical reaction that RELEASES energy - Energy is released from the system - Has a NEGATIVE DeltaG DeltaG = -436 J/mol - Bond BREAKING (because when we break the bonds that hold the molecules together we release energy). 3. ACTIVATION ENERGY - Every reaction, weather Endergonic or Exergonic require Activation Energy to occur. - Activation Energy is the minimum amount of energy required to start a chemical reaction. - Energy must be put into a system in order to get a chemical reaction to occur - Activation energy is essentially an energetic barrier that must be overcome for a chemical reaction to proceed forward - Most reactions would not proceed forward because the reactants are stable - Normally most molecules are very stable in their current configuration, and as a result, reactions generally don't move forward so essentially activation energy allows us to open bonding position so our reactants can be brought in together. Ex.: 2H2 + O2 --> 2 H20 We have to destabilize the valence electrons so we can open up a bonding position and allow the hydrogen and the oxygen to bind together. If you don't have the activation energy, they will just remain as two oxygens bonded together and two oxygen bonded together. --> REDOX Reactions - A chemical reaction involving the transfer of one or more electrons from one reactant to another; also called oxidation-reduction reaction. - Many metabolic reaction in the cells also include oxidation and reduction reactions. - Oxidation and Reduction always occur in PAIRS. * If you remove something, you have to put it somewhere. 1. Oxidation reactions occur when: - ELECTRON DONORS get oxidized * they give oxygen and electrons away A. Can signal the removal of oxygen from the compound B. Can signal the removal of electrons from an atom or electron C. EXERGONIC - Bond breaking that release energy *** ELECTRON DONORS - compounds being OXIDIZED - are thought of as ENERGY sources. * when they are oxidized, energy can be released from them. * the energy might be stored in the bond, not exactly in the electrons. Ex.: NADH -> NADH + H+ Oxidized form -> reduced form to oxidize requires energy. To reduce releases energy. - Energy that is released from Redox pairs must be stored in the cell in order for it to be used later in cellular functions. * most energy is stored in Phosphorylated compounds -->Reduction reactions occur when: - ELECTRON ACCEPTORS get reduced * they accept hydrogen and electrons 1. There is the addition of hydrogen 2. There is the addition of electrons 3. ENDERGONIC - Bond forming. Require energy --> Energy storage - Energy that is released from Redox pairs must be stored in the cell in order for it to be used later in cellular functions. * most energy is stored in Phosphorylated compounds, such as ATP, Easter Bonds and Anhydride bonds - ATP * ATP is phosphorylated because it starts as ADP+P --> ATP * the bond required to hold the 3rd Phosphate is very high in energy. - Easter Bond * forms between alcohols and acids * found in lipids - hold the glycerol heads to the fatty acids chains. * Ex. Glucose 6-Phosphate and ATP - Anhydride * Stabilizes by resonance. * found in Benzine rings - 6 carbons joined together by double and single bonds. The double and single carbons that alternate are referred to as resonance * Ex.: Phosphoenolpyruvate, ATP and actyl phosphate Phosphate bonds are higher in energy than the average covalent bond in a cell

Chemoorganotroph

- Fungi, protozoa and most bacteria - are organisms that obtain its energy from the oxidation of organic compounds. - Have the ability to oxidize chemical bonds found in organic compounds. - The primary organic compound used is CARBOHYDRATES.

Electron Microscope

- Generally used to see bacteria, viruses, internal cellular structures, molecules and and large ATOMS. - Two kinds: 1. Transmission Electron Microscope (TEM) *Because the vacuum and slicing of the specimens are required, transmission electron microscopes cannot be used to study living things. - Specimens prepared for electron microscopy must be dry because water vapor from a wet specimen would stop an electron beam. - Transmission Electron Microscopy requires that the desiccated sample also be sliced very thin, generally before staining. - Specimens for Scanning electrons microscopy are COATED, not stained. - Stains used for transmission Electron Microscopy are not colored dyes but instead chemicals containing atoms of heavy metals, such as LEAD, OSMIUM, TUNGSTEN, and URANIUM, which absorb electrons. * Electron dense stains may bind to molecules within specimens, or they may stain the background. The latter type of negative staining is used to provide contrast for extremely small specimens, such as viruses and molecules. * Stains for Electron Microscopy can be general in that they stain most objects to some degree, or they may be highly specific. For example, OSMIUM TETROXIDE (OsO4) has an affinity for lipids and is thus used to enhance the contrast of membranes. Electron-dense stains can also be linked to antibodies to provide an even greater degree of staining specificity because antibodies bind only to their specific target molecules. 2. Scanning Electron Microscopes (SEM) *Whole specimens can be observed because sectioning is not required. However, it magnifies onl the external surface of a specimen and, like TEM, it requres a VACUUM and thus can examine only dead organisms.

light

- How can it effect bacterial growth? • Some bacteria, like plants, use light as an energy source (autotrophs- phototrophs) - What kind in particular? * UV LIGHT • Most non photosynthetic bacteria are damaged by contact with light • Ultraviolet (UV) light rays are BACTERICIDAL - kills bacteria (such as the ones in the googles or in plaque in the teeth)

Physical characteristics

- Many physical characteristics are used to identify microorganisms. 1. MORPHOLOGY - Scientists can usually identify protozoa, fungi, algae, and parasitic worms based solely on their shape (morphology) 2. PHYSICAL APPEARANCE - Scientists can also use the physical appearance of a bacterial colony (a group of bacteria that has arisen from a single cell grown on a solidi laboratory medium) to help identify microorganisms. - As we have discussed, stains are used to view the size and shape of individual bacterial cells to show the presence or absence of identifying features such as endospores and flagella. - Linnaeus categorized prokaryotic cells into two genera based on two prevalent shapes: 1. COCCUS - Spherical prokaryotes 2. BACILLUS - rod-shapped cells * however, subsequent studies have revealed vast differences among many of the thousands of spherical and rod-shaped prokaryotes, and thus visible characteristics alone are not sufficient to classify prokaryotes. Instead, taxonomists rely primarily on genetic differences as revealed by metabolic dissimilarites and, more and more frequently, on DNA sequences that code for subunits of rRNA.

Explain the metabolic pathways that bacteria can use (e.g. aerobic respiration, glycolysis, fermentation)

- Metabolism is the sum of all metabolic reactions in a cell - It envolves: 1. ANABOLISM - is the formation of more complex products - Ex. taking a monomer and joining it to make a macromolecule. - ENDERGONIC * Because it requires the input of energy in order to form the bonds between the small subunits to make something larger 2. CATABOLISM - is the breaking of complex products into simpler products. - EXERGONIC *Because you break bonds in order to turn something large into small peaces. - Catabolism can be used to release free energy from substrates which can be transferred to energy storing molecules, such as ATP - two types of metabolism: Fermentation and Respiration. 1st) GLYCOLYSIS - DOES NOT REQUIRE OXYGEN! A metabolic process that breaks down carbohydrates and sugars through a series of reactions to either pyruvic acid or lactic acid and release energy for the body in the form of ATP. - Every metabolic reaction (fermentation or respiration) begins with glycolysis. - Glycolysis = spliting glucose. ---> INVESTMENT STAGE (endergonic phase) 1. Begins with glucose 2. + Add ATP (Activation Energy) 3. = Glucose 6-P (the first intermediate). *Also happens when during group translocation - when the cell brings the glucose across the plasma membrane, this step can occur automatically, giving us G6P. 4. G6P gets rearranged into Fructose 6-P * no bonds were broken or made - they were only rearranged, going from a 6 sided sugar to a 5 sided sugar. * Glucose 6P and Fructose 6-P are ISOMERS. Same pieces, different shapes. 5. + Add a 2nd ATP. * it gets phosphorylated 6. G6P + ATP turns into Fructose 1, 6 Biphosphate * it is gonna have those 2 phosphate groups on it --> LYSIS STAGE (exergonic phase) - called Lysis stage because this is when we are going to break bonds for the first time 7. Fructose 1, 6 Biphosphate gets broken down into two 3-carbon compounds: G3P and DHAP. * One big molecule is broken into two smaller molecules: Glyceraldehyde-3-P (G3P) and Dihydroxyacetone-P (DHAP). ** when we break Fructose 1, 6P, we release energy ---> ENERGY- CONSERVING STAGE 8. Energy is released from the broken bond that split Fructose 1. 6P. 9. The energy released will be used later on in a REDOX REACTION - used to reduce NAD+, STORING energy into NADH. *NAD+ gets reduced using the energy that was released when Fructose 1, 6 Biphosphate got broken down into Glyceraldehyde-3-P and Dihydroxyacetone-P 10. Glyceraldehyde-3-P gets converted (OXIDIZED) into the 1, 3P BisphosphoglycerIC Acid. *Another phosphate is added 11. 2 ATPs are gets created 12. Also 3-P-Glycerate is created 13. 3-P-Glycerate gets rearranged into 2-P-Glycerate 14. 2-P-Glycerate gets rearranged into Phosphoenolpyruvate. 15. Phosphoenolpyruvate gets rearranged into PYRUVATE. *2nd 2 ATPs generated while rearranged Phosphoenolpyruvate into PYRUVATE --> PYRUVATE IS THE STARTING POINT FOR ALL METABOLIC REACTIONS ---> 2 ATPs used during the INVESTMENT STAGE and 4 ATPs CREATED during the ENERGY-CONSERVATION PHASE = NET 2TP + 2 NADH * Next step: FERMENTATION or OXIDATIVE PHOSPHORYLATION 2nd EITHER). FERMENTATION - It is one kind of metabolism - is substrate level phosphorylation, because the substrate is directly phosphorylated. That is, a phosphate group is added to it. - Substrate becomes the electron donor and also the final terminal electron acceptor - DOES NOT REQUIRE OXYGEN! - Once glycolysis has been completed and PYRUVATE has been formed, the cell has some choices to make metabolically. If the cell cannot continue to oxidize Pyruvate through cellular respiration, it has to have a some mechanism through which it can regain the NAD+ used during Glycolysis. - During the Energy Conservation phase we took NAD+ and reduced it to NADH. The cell needs to take that NADH and OXIDIZE it back to NAD+ - So FERMENTATION PATHWAYS will allow a cell to form alternate source of NAD so it can be supplied with constant source of NAD+ for glycolysis. * Cannot be obtained simply using glycolysis and Krebs cycle - Fermentation pathways provide cells with alternate source of NAD+ - Fermentation is essentially a partial oxidation of the remainder of the sugar (or other metabolites). This partial oxidation is going to release additional energy that is in the molecule. It also provides the cell with an organic molecule from within the cell as final electron acceptor. - it gives us a place to put those electrons that we used to reduce NAD+. -Pyruvate that is formed from glycolysis can be used to form several different products (different fermentation pathways) a. ALCOHOL FERMENTATION (mostly yeast) • The Pyruvate created during glycolysis gets Decarboxylated, meaning a carboxyl group gets removed (H-O-C=O). - Decarboxylation of pyruvic acid turns it into acetaldehyde • Acetaldehyde is reduced to ethanol (the H+ used to reduce it comes from NADH) NADH --oxidized --> NAD + H --Hydrogens are put into--> Acetylaldehyde = ETHANOL b. ACIDIC FERMENTATION (bacteria) • Pyruvic acid is reduced into lactic acid (lactate) • H+ comes from NADH NADH --oxidized --> NAD + H --Hydrogens are put into--> = LACTIC ACID. • Some bacteria undergo HOMOlactic fermentation (only lactic acid is made) • Other bacterias undergo HETErolactic fermentation (lactic acid, acetic acid, CO2) * As this bacteria ferment, they are making acid products, so they are going to allow us to drop the pH in the test tube that they are growing in. So we can look for this decline in pH by using a pH indicator called Phenol Red. We can find it in many of carbohydrate fermentation media that we are going to look at. If the bacteria can ferment the sugar, it can produce acid. If it produce acid, it will change the pH in the tube. Also the Heterolactic fermentors can produce CO2, which is gas. * So we can look for the ability to fermentation by the production of acid or gas c. MIXED ACID FERMENTATION - Many bacteria can do Mixed Acid Fermentation • Pyruvate is enzymatically convert to a number of acids- acetic, lactic, succinic and formic acid. * The result will be a drop of pH in the test tubes. ---> FERMENTATION ALSO ALLOWS YOU TO PRODUCE 2 ATPs (which increases glycolysis rate, or quantity, allowing for more robust growth of the bacteria 1. ---> Because Fermentation doesn't not require oxygen, FACULTATIVE ANAEROBES can use this in order to grow and survive when oxygen is absent from their environment. 2. ---> Bacterial cells that are ANAEROBES (the ones that grow without oxygen) can use fermentation as their metabolic pathways. 3. ---> Because fermentation doesn't require oxygen, that means we can have bacteria that colonize in ANAEROBIC environments. * so while most types of species would not be able to survive in anaerobic environments, bacterial cells can if they can utilize FERMENTATION. OR 2nd). CELLULAR RESPIRATION (or OXIDATIVE PHOSPHORYLATION) - It is another kind of metabolic reaction - is oxidative phosphorylation • Substrate is electron donor and oxygen (if it is aerobic) is the terminal acceptor. ***If anaerobic, something other than oxygen is the terminal electron acceptor. • Uses proton motive force to generate ATP - Pyruvic acid, that was made in glycolysis, can continue to be oxidized in order to release more energy - Instead of fermentation, some bacteria are able to make Oxidative phosphorylation, which uses oxygen to continue to break glucose down (break more bonds) and release more energy from glucose. - You can get more energy per glucose molecule • Pyruvate is oxidized and CO2 (gas) is released • Coenzyme A is attached • Acetyl CoA is formed and sent to the Kreb's cycle for further oxidation. - it also creates NADH. - KREB'S CYCLE - or Citric Acid Cycle. The Kreb's cycle is a series of oxidative reactions that allows us to further break down Acetyl CoA. It begins with the cleavage of CO2 - so we are going to release more CO2 molecules. • The Kreb's cycle goal is to move electrons of the Acetyl CoA and use them to reduce energy intermediates. --> Acetyl CoA will act as an ELECTRON DONOR and those oxidative reactions to make it an Electron donor will be used to reduce the energy intermediates NADH and FADH2. • The energy intermediates NADH and FADH2 provide energy for the proton motive force- electron transport chain ( which is where the ATP is ultimately made) 1. Oxaloacetate is joined to Acetyl CoA made from the pyruvic acid. * We use the Oxaloacetate and then we use the Kreb's cycle to remake it over and over again in a cycle. 2. Acetyl CoA is the starting product for the Kreb's Cycle. 3. Oxaloacetate plus Acetyl Coa = forms the first product, the Citrate, or Citric Acid. 4. Citrate is rearranged into Isocitrate. 5. Isocitrate is Oxidized into Alpha-Ketoglutarate and CO2 is released. * By breaking the Isocitrate and releasing CO2, energy was created, and stored, reducing NAD+ into NADH. 6. Alpha-Ketoglutarate is oxidized into Succinyl CoenzymeA, and CO2 is released again. * By breaking the Alpha-Ketoglutarate and releasing CO2, energy was created, and stored, reducing NAD+ into NADH again. 7. Succinyl CoenzymeA gets rearranged into Succinate and as a result, ATP is produced. 8. Succinate gets rearranged into Fumarate * By rearranging the Succinate, energy was created, and stored, reducing FAD into FADH2. 9. Fumarate gets rearranged into Malate. 10. Malate gets rearranged into Oxaloacetate again. * By rearranging the Malate, energy was created, and stored, reducing NAD+ into NADH again. ** This last energy created is brought into the new cycle, and used to join the Oxaloacetate that was just created with the new Acetyl CoA that is entering and starting the new cycle. RESULTS: - Energy storage in the form of: * NADH * FADH2 * ATP * And the release of CO2 REDUCED FORMS ARE THE ONES THAT STORE ENERGY ON THEM! - NADH and FADH created during Kreb's cycle are sent to the ETC - Electron Transportation Chain. --> ETC (ELECTRON TRANSPORTATION CHAIN) - NADH and FADH2 created during the Kreb's cycle are OXIDIZED and sent to the ETC - Electron Transportation Chain. * when we oxidize the NADH and FADH2 they will release energy and the energy in the carriers will be used to power proton pumps in order to create a GRADIENT. - The proton pumps will allow us to pump Hydrogen into the extracellular space. * we will form a gradient of protons on the outside of the cell (POSITIVELY CHARGED) that will be greater in the outside than inside the cell ( NEGATIVELY CHARGED). - By creating that gradient, we will actually make an Electrochemical Potential. * This energy can be used to do a number of work, including Flagella rotation, as well as to make ATP. * GRADIENT: low concentration of Hydrogen Ions inside and high concentration of Hydrogen ions outside the cell 1. COMPLEX 1 - Energy from NADH is oxidized and the protein Complex 1 is used to pump the Hydrogen across the membrane, turning back into NAD+. 2. COMPLEX 2 - Energy from FADH2 is oxidized and the protein Complex 2 is used to pump the Hydrogen across the membrane, turning back into FAD+. 3. COMPLEX 4 - CYTOCHROME OXIDASE - Cytochrome oxidase (Complex IV) accepts the electrons from cytochrome c and allows us to remove H+ from the solution. - We have to do something with the electrons that are coming out of the Cytochrome C and into complex 4. * If we have an AEROBIC bacteria, that is, one that uses oxygen, then the electrons that come out of complex 4 will be used to O2 is reduced to form water. (The aerobic bacteria uses the O2 that, when it joins with the H, turns into H2O) ** If the bacteria ACCIDENTLY do not perform this process correctly, than this improper reduction forms superoxides. SUPEROXIDE ARE VERY TOXIC because they can destroy the plasma membrane. As a result of this, all aerobic bacteria has to have ENZYMES that help break down the superoxides. - Aerobes need enzymes to counteract the effects of the superoxides to break them down. • The two major enzymes are: 1. SOD - Super Oxide Dismutase 2. catalase ---> If a bacteria does not have CYTOCHROME, CATALASE or SOD, it is considered ANAEROBIC (they would need to live in an environment away from oxygen because oxygen is toxic). ---> EXCEPTION: Streptococcus species are an exception, they lack catalase and many do not have cytochromes. However, they are still considered aerobic and can live in an environment with oxygen in it. Hydrogen carriers reduce electron carriers (Complex 1 FMN, Fe/S) • Protons are forced to the exterior of the plasma membrane • Note the change in the Redox potential of the membrane as protons are forced to the exterior of the cell membrane • Creates an electrochemical potential • Potential energy in the pmf is used to phosphorylate ADP • pmf can also be to do other types of work, such as ion transport and flagellar rotation - Now that we have a proton gradient with high concentration of Hydrogen atoms outside of the cell and low concentration inside the plasma membrane, this gradient concentrate has a lot of potential energy in it. The cell needs to translate this potential energy into an useful form. In order to turn it into useful form: - The energy from the pmf is dissipated and captured in an enzyme called the ATP synthase. * ATP synthase (a protein embedded in the plasma membrane) can take the energy that is in the gradient and turn it into ATP, which is a useful source of ATP synthase. • H+ diffuse (from high to low) through the F0 ion pump (inside the ATP Synthase) which causes rotation. • F0 is coupled to F1 (through the stator). Every time an H+ is pumped through the F0, F0 rotates and F1 also rotates. • This causes the enzyme to move between an open conformation which accepts ADP and P and a closed conformation which phosphorilates ADP + P, binding them and generating ATP. ---> Some microbes, like yeast, can do both RESPIRATION and FERMENTATION depending on the presence of O2

Confocal Microscopes

- Microscopes that use laser to illuminate fluorescent chemicals in a thin plane of a specimen.

Negative (Capsule) Stains

- Most dyes used to stain bacterial cells, such as Crystal Violet, Methylene Blue, Malachite Green, and Safranin are basic dyes. - These dyes stain cells by attaching to NEGATIVELY charged molecules within them - ACID DYES, by contrast, are repulsed by the negative charges on the surface of cells and, therefore, do not stain them. Such stains are called NEGATIVE STAINS because they stain the BACKGROUND and leave CELLS COLORLESS. - EOSIN and NIGROSIN are examples of acidic dyes used for NEGATIVE staining. ** Negative stains are used primarily to revel the presence of negatively charged bacterial capsules. Therefore, they are also called CAPSULE STAINS, Encapsulated cells appear to have a HALO surrounding them.

Differential Staining

- Most stains used in Microbiology are DIFFERENTIAL STAINS, which use more than one dye so that different cells, chemicals, or structures can be distinguished when microscopically examined. - They are: 1. GRAM STAIN 2. ACID FAST STAIN 3. ENDOSPORE STAIN 4. GOMORI METHENAMINE SILVER STAIN 5. HEMATOSYLIN 6. EOSIN STAIN

Chemolithotrophs

- Nitrifying bacteria (found in plants) - organisms that obtain energy from the oxidation of inorganic compounds. - They oxidize chemical bonds in inorganic compounds. - They are able to fix nitrogen from the atmosphere.

Describe the oxygen requirements of bacteria (e.g. strict aerobe, microaerophilic, etc)

- OXYGEN PROCESSING - Bacteria needs carbohydrates, special growth factors and oxygen (or lack of it) to grow. - All aerobes and facultative anaerobes use oxygen for growth and metabolism • Oxygen reduction can generate free radicals which are highly damaging. * incomplete reduction of oxygen at the end of CELLULAR RESPIRATION: when the electron transport chain is putting those electrons on to the oxygen in order to make water, if this doesn't occur correctly, it can produce : --> Superoxide anions, peroxides, and hydroxyl radicals • Cells use enzymes (such as SOD and CATALASE) to neutralize these free radical and superoxides * SOD (Superoxide Dismutase) will transform the superoxide into oxygen and also join the oxygen to the hydrogen in order to make hydrogen peroxide. ** Hydrogen peroxide can be quite damaging to cells, so the second enzyme CATALASE takes the hydrogen peroxide and breaks it back into oxygen and water, which are harmless • Without these enzymes, the superoxides and oxides formed by improper processing at the end of the ETC become toxic to cells. - CATALASE TESTING - • Because catalase is found in most aerobic and facultative anaerobic bacteria it can be used for identification purposes (phenotypic) • Useful in differentiating Staphylococci and Micrococcacae from Streptococci and Enterococci • Streptococci are facultative anaerobes, why can the catalase test be used tell them apart from Streptococcus? - Note: never do this on blood agar! RBCs have catalase in them and give false positive results 1. AEROBE Oxygen requirement: - uses oxygen to grow Ex.: Mycobacterium tuberculosis - Which growns in the lungs and causes TB (Tuberculosis) 2. OBLIGATE AEROBE -Oxygen requirement: must have oxygen to grow Ex.: Nocardia asteroides- causes infections of the heart valves 3. FACULTATIVE ANAEROBE - Oxygen requirement: is actually an AEROBE that grows best in the presence of oxygen, however they are facultative so if the oxygen is removed from the environment, they can still survive and grow (not very well) in the absence of oxygen. Ex.: Staphylococcus aureus 4. ANAEROBE Oxygen requirement: - grows in the absence of oxygen * If oxygen is present, it is toxic because the anaerobes don't have SOD and CATALASE to deal with the Superoxide anions, peroxides, and hydroxyl radicals that are made after incomplete reduction of oxygen at the end of CELLULAR RESPIRATION (when the electron transport chain is putting those electrons on to the oxygen in order to make water) Ex.: Bacteroides fragilis 5. OBLIGATE ANAEROBE Oxygen requirement: - can not use oxygen and DIES in its presence * If oxygen is present, it is toxic because the anaerobes don't have SOD and CATALASE to deal with the Superoxide anions, peroxides, and hydroxyl radicals that are made after incomplete reduction of oxygen at the end of CELLULAR RESPIRATION (when the electron transport chain is putting those electrons on to the oxygen in order to make water) Ex.: Clostridium botulinum and Clostridium. perfringenes 6. AEROTOLERANT ANAEROBE Oxygen requirement: - An organism that is an ANAEROBE, meaning that it grows best when oxygen is not present, however it is AEROTOLERANT, meaning it could still survive in the presence of oxygen although it wouldn't grow very well Ex.: Lactobacillus sp. 7. MICROAEROPHILE Oxygen requirement: - microaerophilic: require SMALL amounts of bacteria - are bacteria the require oxygen to grow, but can only tolerate it at concentrations lower than that found in atmospheric air Ex.: Campylobacter jejuni normally colonizes poultry digestive tracts. Also a leading causes of food borne illness in the US * It is gram NEGATIVE and helical

Resolution

- Or RESOLVING POWER - is the ability to distinguish two points that are close together. - It depends on: 1. The wavelength of the electromagnetic radiation 2. The NUMERICAL APERTURE of the lens, which refers to the ability of lens to gather light. Resolution= (0.61 x wavelength)/Numerical Aperture

Bacteria

- PROkaryotic - Has FEW FREE ORGANELLES bound with phospholipid membranes - Has GLYCOCALYX - MOTILITY present in some - Some have FLAGELLA, each composed of basal body, hook and filament; flagella ROTATE - No CIlia - Some have FIMBRIAE or PILI - Does Not have Hami - Most have CELL WALL , COMPOSED OF PEPTIDOGLYCAN - All have CYTOPLASMIC MEMBRANE - All have CYTOSOL - All have INCLUSIONS - some have ENDOSPORES - Have small 70S RIBOSOMES for PROTEIN SYNTHESIS - CHROMOSOMES are commonly single and circular. - Some have CYTOSKELETON - to SHAPE in prokaryotes; - NO centromere

Contrast

- Refers to differences in intensity between two objects or between an object and its background. - Important in determining resolution. * Most microorganisms are colorless and have very little contrast whether one uses light or electrons. one way to increase the contrast between microorganisms and their background is to stain them. * The use of LIGHT that is in PHASE - that is, in which all of the waves' crests and troughs are aligned - can also enhance contrast.

Thioglycollate medium

1. Type of Media: - is a differential liquid medium 2. Differentiates for: - classifies oxygen requirements for the bacteria we are growing. 3. Results: • The area where growth (cloudiness) occurs determines the oxygen requirement 4. Notes: In the picture: 1. Strict (Obligate) Aerobe *Growth only occurs on the very top of the tub. 2. Facultative Anaerobe * Requires oxygen but can still survive in the absence * The haviest growth is in the top, but you still see some throught the tube 3. Aerotolerant Anaerobe * Does not require oxygen, but it can survive in the presence of the oxygen. 4. Strict Anaerobe * Growth only occurs on the bottom of the tube.

Endospore Stain

- Some bacteria - notably those of the genera BACILLUS and CLOSTRIDIUM, which contain species that cause such diseases as Antrax, Gangrene and Tetanus - produce ENDOSPORE - these dormant, highly resistant cells form inside the CYTOPLASM of the BACTERIA and can survive environmental EXTREMES such as desiccation, heat, and harmful chemicals. - Endospores cannot be stained by normal staining procedures because their walls are practically IMPERMEABLE AT ROOM TEMPERATURE. 1. the SCHAEFFER-FULTON ENDOSPORE STAIN uses heat to drive the primary stain, MALACHITE GREEN, into the endospore. After cooling, the slide is decolorized with water and conterstained with SAFRANING. This staining procedure results in GREEN-STAINED ENDOSPORES and RED-COLORED vegetative cells. 2. HISTOLOGICAL STAINS - Laboratory technicians use two popular stains to stain histological specimens, that is, tissue samples: a. GOMORI METHENAMINE SILVER (GMS) STAIN - commonly used to screen for the presence of fungi and the locations of carbohydrates in tissues. b. HEMATOXYLIN AND EOSIN (HE) STAIN - which involves applying the basic dye hematoxylin and the acidic dye eosin, is used to delineate many features of histological specimens, such as the presence of cancer cells.

Predict what will happen to a cell in solutions of various tonicities.

- Stains make microorganisms and their parts more visible because stains increase contrast between structures and between a specimen and its background. - Most microorganisms are colorless and have very little contrast whether one uses light or electrons. one way to increase the contrast between microorganisms and their background is to stain them. - Stains make microorganisms and their parts more visible because stains increase contrast between structures and between a specimen and its background. - It simply means COLORING SPECIMENS WITH STAINS (which are also called DYES). - Desiccation (drying) and fixation kill the microorganisms, attach them firmly to the slide, and generally preserve their shape and size. It is important to smear and fix the specimens properly so that they are not lost during staining. - Are usually SALTS - SALTS are composed of a CATION+ and an ANION- - At least onf of the two ions in the molecular makeup of dyes is colored, this colored portion of a die is known as the CHROMOPHORE. Chromophores bind to chemicals via covalent, ionic, or hydrogen bonds. *Ex.: Methylene blue chloride is composed of a cat-ion chromophore, methyle1ne blue, and a chloride anion. Because methylene blue is positively charged, it ionically bonds to negatively charged molecules in cells, including DNA and many proteins. In contrast, anionic dyes, for example, eosin, bind to positively charged molecules such as some amino acids. 1. ACIDIC DYES - Anionic chromophores are also called acidic dyes because they stain alkaline structures and work best in acid (low pH) environments. 2. BASIC DYES - Positively charged, cation chromophores are called basic dyes because they combine with and stain acidic structures; further, they work best under basic (higher pH) conditions. **In microbiology, BASIC dyes are used more commonly than acidic dyes because most cells are negatively charged. 1. PREPARING THE SPECIMENS FOR STAINING - Place the microorganisms on a microscope slide and firmly attach them to it: a. Make a SMEAR *If the organisms are growing in a liquid, the microscopist spreads a small drop of the broth across the surface of the slide. * If the organisms are growing on a solid surface, such as an agar plate, they are mixed into a small drop of water on the slide. b. AIR DRY the Smear c. FIX it to the slide by: --> HEAT FIXATION - Developed by Robert Koch. * The slide is gently heated by passing the slide, smear up, through a flame. --> CHEMICAL FIXATION - involves applyig a chemical such as METHYL ALCOHOL to the smear for 1 minute. 2. PRINCIPLES OF STAINING: * Dyes used as microbiological stains for light microscopy are usually SALTS. --> BASIC SALTS In microbiology, BASIC DYES are used more commonly than acidic dyes because most cells are negatively charged. --> ACIDIC SALTS a. SIMPLE Staining: - Soak the smear in the dye for 30-60 seconds and then rinsing off the slide with water. *Composed of a single basic dye, such as CRYSTAL VIOLET, SAFRANIN or METHYLENE BLUE. - Carefully blotting the slide dry - Observe the smear under the Microscope OOOOORRRRR b. DIFFERENTIAL Staining:

Glycocalyx

- Sweet Cup - gelatinous, sticky substance that surrounds the outside of the cell. - May be composed of: 1. POLYSACCHARIDES 2. POLYPEPTIDES 3. BOTH. - These chemicals are produced inside the cell and are extruded onto the cell's surface. - Two Types: 1. CAPSULE - When the glycocalyx of a bacterium is composed of organized repeating units of organic chemicals firmly attached to the cell's surface, the glycocalyx is called capsule. * The chemicals in many bacterial capsules can be similar to chemicals normally found in the body, preventing bacteria from being recognized or devoured by defensive cells of the host. 2. SLIME LAYER - A loose, water-soluble glycocalyx * Are often sticky and provide one means for bacteria to attach to surfaces as BIOFILMS, which are aggregates of many bacteria living together on surface. Ex. Oral bacteria colonize the teeth as a biofilm called PLAQUE. * Bacteria in a dental biofilm can produce ACID and cause DENTAL CARIES (CAVITIES). - Protect cells from drying (DISSECATION) - Plays a role in the ability of pathogens to survive and cause disease. - When the glycocalyx of a bacterium is composed of organized repeating units of organic chemicals firmly attached to the cell's surface, the glycocalyx is called capsule. - The chemicals in many bacterial capsules can be similar to chemicals normally found in the body, preventing bacteria from being recognized or devoured by defensive cells of the host. * Ex. The Capsules of Streptococcus Pneumoniae and Klebsiella Pneumoniae enable these prokaryotes to avoid destruction by defensive cells in the respiratory tract and do cause pneumonia. Unencapsulated strains of these same bacterial species do not cause disease because the body's defensive cells destroy them.

Eukaryotic cell

- TRUE NUCLEUS - Have a NUCLEAR ENVELOPE surrounding their DNA, forming a NUCLEUS. - Also has mebmbrane-bound ORGANELLES - specialized structures that act tiny organs to carry on the various functions of the cell. - Larger than Prokaryotes - More complex than Prokaryotes - EUkaryotic - Has various types of FREE ORGANELLES bound with phospholipid membranes present in all: 1. ER 2. Golgi Bodies 3. Lysosomes 4. Mitochondria 3. Chloroplasts. * Also has PEROXISOMES, VACUOLES AND VESICLES. - Has GLYCOCALYX in some ANIMAL cells - MOTILITY present in some - some have Flagella, Cilia or Pseudopods. - Some have FLAGELLA or CILLIA, composed of a 9+2 arrangement of MICROTUBULES; Flagella and Cilia undulate. - CILIA present in some - Do not have Fimbriae or Pili - Does Not have Hami - CELL WALL present in plants - All have CYTOPLASMIC MEMBRANE - All have CYTOSOL - some have INCLUSIONS - Do not have Endospores - Have LARGE 80S RIBOSOMES in Cytosol and on ER, and smaller 70S in Mitochondria and Chloroplasts - all for PROTEIN SYNTHESIS - CHROMOSOMES are commonly LINEAR and more than one chromosome per cell. - All have CYTOSKELETON - for SUPPORT, CYTOPLASMIC STREAMING, and ENDOCYTOSIS in Eukaryiotes - some ANIMALS have CENTROSOME.

How is blood agar differential?

1. Type of Media: - DIFFERENTIAL MEDIA 2. Differentiates for: • When streaked on Blood Agar, many species of bacteria cause hemolysis - i.e., destruction of the erythrocytes (and hemoglobin) in the medium * Allows us to differentiate the microbes abilities to cause hemolysis a. Alpha hemolysis • Streptococcus mitis growing on Blood Agar; some greenish zones of alpha hemolysis are visible * Can only partially distroy the red blood cells. They live behind a discolored green. b. Beta hemolysis • Enterococcus durans growing on Blood Agar, the light to clear zones can be seen around the colonies * The visible clear halo results because they can completely syse and distroy the red blood cells in the plate. c. Gamma hemolysis * The bacteria grows but does not distroy the red bloodcells, meaning NO hemolysis at all. 3. Results: 4. Notes:

Bright Field Microscope

- The most common microscopes in which the background (or field) is illuminated. - They are two kinds: 1. SIMPLE MICROSCOPES - a Bright Field Microscope - Such as Leeuwenhoek's in 1674 - contains a SINGLE magnifying lens - Similar to a MAGNIFYING GLASS 2. COMPOUND MICROSCOPES - a Bright Field Microscope - Uses a series of lenses for magnification - Wasn't until 1830 that scientists developed compound microscopes that exceeded the clarity and magnification of Leeuwenhoek. - Magnification is achieved as light rays pass through a specimen and into an OBJECTIVE LENS (4x, 10x, 40x, 100x) - An objective lens BENDS the light rays, which the pass up through one or two OCULAR LENSES. (10x) - TOTAL MAGNIFICATION = Ocular lens x Objective Lens - Modern compound microscopes also have a CONDENSER LENS --> OBJECTIVE LENS - Series of lenses that not only create a magnified image but also are engineered to reduce aberrations in the shape and color of image. - Most light microscopes have 3 or 4 objective lenses mounted on a REVOLVING NOSEPIECE. - The objective lenses on a typical microscope are 1. Scanning objective lens (4X) 2. Lower power objective lens (10x) 3. High power lens or Hight Dry objective lens (40x) 4. Oil Immersion Objective Lens (100x) *An oil immersion lens increases not only magnification but also resolution. * Light refracts as it travels from air into glass and also from glass into air; therefore, some of the light passing out of a glass slide is bent so much that it bypasses the lens. Placing IMMERSION OIL (Historically cedarwood oil; today more commonly a synthetic oil) between the slide and an oil immersion objective lens enables the lens to capture this light because light travels through immersion oil at the same speed as through glass. Because light is traveling at a uniform speed through the slide, the immersion oil, and the glass lens it does not refract. * Immersion oil increases the numerical aperture, which increases resolution, because more light rays are gathered into the lens to produce the image. *Obviously, the space between the slide and the lens can be filled with oil only if the distance between the lens and the specimen called the WORKING DISTANCE is small --> OCULAR LENSES - the lenses closest to the eyes. - They can be MONOCULAR or BINOCULAR. - They magnify the image created by the objective lens is 10x. --> TOTAL MAGNIFICATION - of a compound microscope is determined by multiplying the magnification of the objective lens by the magnification of the ocular lens. Total magnification = 10x ocular X 10x low-power objective = 100x Total magnification = 10x ocular X 100x immersion oil objective = 1000x

Fluorescence Microscopes

- Use invisible ultraviolet light to cause specimens to radiate visible light, a phenomenon called FLUORESCENCE. - It is also used in a process called IMMUNOFLUORESCENCE. First, fluorescent dyes are chemically linked to Y-shaped immune system proteins called ANTI-BODIES. Antibodies will bind specifically to complementary-shapped ANTIGENS, which are portions of molecules that are present, for example, on the surface of microbial cells. When viewed under UV light, a microbial specimen that has bound dye-tagged antibodies become visible.

Fluorescent stains

- Use invisible ultraviolet light to cause specimens to radiate visible light, a phenomenon called FLUORESCENCE. - It is also used in a process called IMMUNOFLUORESCENCE. First, fluorescent dyes are chemically linked to Y-shaped immune system proteins called ANTI-BODIES. Antibodies will bind specifically to complementary-shapped ANTIGENS, which are portions of molecules that are present, for example, on the surface of microbial cells. When viewed under UV light, a microbial specimen that has bound dye-tagged antibodies become visible.

Simple Staining

- Used to determine size, shape, and arrangement of cells. - Composed of a single basic dye, such as CRYSTAL VIOLET, SAFRANIN or METHYLENE BLUE 1. Soak the smear in the dye for 30-60 seconds and then rinsing off the slide with water. 2. Carefully blotting the slide dry 3. Observe the smear under the Microscope

osmotic pressure

- What bacterial structure would contribute to osmotolerance? --> Cell walls - Bacteria is usually hypertonic on the inside, as a result, water is going to rush into their cells. We know that the cell walls are made to prevent the cell from lysing in mildly hypertonic environment, but if the environment is very hypertonic, the cell will loose water to the environment. Two types: 1. Halophiles 2. Osmotolerant

biofilm

- What is? - How do they form? 1. Biofilms begin with colonization of a surface of "something" by binding to the surface, using extracellular matrix (like the glycocalyx), fimbriae and or pili - They are free floating, called PLANKTONIC BACTERIA. 2. When the Planktonic bacteria cell attaches to the surface, the cells that gather begin to produce extracellular polymeric substances (EPS), * EPS will allow the bacteria to adhere more strongly to the surface 3. Once the bacteria start to produce EPS, it starts to change phenotypically. The planktonic bacteria have a different phenotype than the bacteria that is attached to the surface because secretion of EPS starts to triggers a change in the types of proteins that the bacteria is synthesizing. The proteome, or the type of proteins that they express, are different whether they are planktonic or whether they are attached to the surface. * Produces phenotypically distinct bacteria 4. Biofilms can be made of many species of microbes (including protozoa and fungi attached to the same surface, and they work together in order to improve the survival of the entire biofilm ) * Can be very complex. 5. When it is time to reproduce, some of the bacteria in the middle of the biofilm will revert back to the free- planktonic stage and be released back into the environment where they can attach to new surfaces and create new biofilms. - How does it benefit the bacteria that are part of it? - How does it tolerate the presence of antibiotics? • Can be antibiotic resistance because of reduced penetration. * The thicker the biofilm, the more EPS is being made, triggering the change in the type of proteins being synthesized. * Antibiotic resistance in 4 stages: a. Slow penetration b. Stress response c. Altered microenvironment d. Persisters - Why is this significant to a medical provider? * Biofilms can provide the bacteria with pathogenic advantage, which helps it to go through the stages of Biofilm Tolerance in order to survive. * It can cause plaque, which is hard to remove * It can inhibit wound healing because the epithelial cells cannot repair themselves so they are unable to stitch back together to repair and the bacteria itself will prevent any type of topical antibiotic to penetrate in to clear up the bacterial infection as well as it will prevent immune cells to reach the site and clear up the infected wound as well

Virus

- are ACELLULAR and generally LACK rRNA. - Virologists do classify viruses into families and GENERA, but higher taxa are poorly defined for viruses.

Predict what would happen if mutations occurred each of the 3 proteins

- have a great effect in bacteria because bacteria only have one single chromossome. - Therefore, if you mutate that gene, the bacteria has to express that gene. Can be: 1. Spontaneous Mutations • Occur because of accidental or spontaneous errors in replication of DNA • Antibiotic resistance in bacteria may be an inherent trait of the organism (e.g. a particular type of cell wall structure) • Spontaneous mutation gives rise to the natural resistance • Acquired resistance can result from mutations (because you are acquiring this from DNA replication: a mutation occurs in the cell and then the cell replicates DNA and divides the one gene that is expressed that is now mutated) • Spontaneous mutation frequency for antibiotic resistance is about of about 10 to the 8th power - 10 to the 9th power • One in every 10to the 8th power - 10to the 9th power bacteria in an infection will develop resistance by mutation • For E. coli: streptomycin resistance is acquired at a rate of 109 when exposed to high concentrations of streptomycin • This seems rare, but the doubling time of E. coli is 20 minutes so you could have over 2x10 to the 157,826 power cells in about 7 hours 2. Induced Mutations • Occur because of mutagens * Mutogens force change in the DNA - Bacteria can be tested to see if it has spontaneous mutation by testing with the AMES ASSAY

Magnification

- is an apparent increase in the size of an object. - Indicated by X (times) - It happens when a beam of radiation refracts (bends) as it passes through a lens. - Curved glass lens refract light, and magnetic fields act as lenses to refract electron beams. - light also bends when it leaves the glass and reenters the air. - Because of its curvature, a lens refracts light rays that pass through its periphery more than light rays that pass through its center, so that the lens focuses light rays on a FOCAL POINT. - Importantly for the purpose of microscopy, light rays spread apart as they travel pass the focal point and produce an enlarged INVERTED IMAGE.

What is a nosocomial infection and where is it acquired.

-> Passive carriers - transfer infectious agents through contact only and are not infected (do not get sick). • Medical and dental personnel that handle heavily contaminated material and transmit them from patient to patient are passive carriers. • Mosquitoes are passive carriers of Plasmodium vivax which causes malaria in humans * they bite an infected person and then they bite a healthy person and then that healthy person is exposed to the sick person's blood, so then this healthy person get sick. --> Associated with NOSOCOMIAL INFECTIONS nosocomial nosocomial infection • Nosocomial infections are acquired in the hospital * Associated with PASSIVE CARRIER • The hospital is a major RESERVOIR for many pathogens • MRSA • Found with compromised hosts (because they are opportunistic infections) • Chain of Transmission (passive carriers) who have direct contact with patients every day, fomites (endoscopes, catheters) are used in procedures • Control of infections (hand washing and compliance) * There are low compliance for hand washing Ex.: Emerging nosocomial infections: 1. Acinetobacter Baumannii - pathogen: • Acinetobacter Baumannii • Pleomorphic (it doesn't have a standard shape - can be anything from a cocci to a rod), gram-negative rod - Reservoir • Natural reservoir is the soil and in the environment • Present in the GI tract of patients in the hospital ** theory is that sick patients have it in their GI tract and are spreading it through fecal matter, however, the hospital staff is not washing hands and spreading it to other immunocompromised patients. • Lives on skin and surfaces (survives for days on surfaces) * Reservoir can be healthy, and still be carrying it for days and transmitting it to immunocompromised patients in the hospital - symptoms - how it is acquired • Healthy individuals are not likely to become infected • Patients with compromised immune systems, surgical wounds, urinary catheters and those on respiratory ventilators are at risk - treatment • Naturally resistant to many common antibiotics - Symptoms? 2. Clostridium Difficile - Nosocomial Infection - pathogen • Clostridium difficile- gram positive, endospore former - symptoms • Produce an enterotoxin that causes symptoms: • Bloating • Diarrhea • Inflammation of colon • Fever - how it is acquired • Can be part of normal flora (colon) in some people * Fecal-oral bacterial route. • Replicates and overruns the colon when the competing normal flora are killed (usually through antibiotic therapy) * It is an endospore former, therefore it will survive when the normal flora get killed by the antibiotics. • Most patients are exposed to the endospores in hospital setting • Fecal contamination, and inadequate disinfection • Endospore * Almost impossible to get rid of. ---> Healthcare workers also act as Passive carriers, transmitting it from patients who have it in their colon to patients that don't have it - treatment

Describe the 3 shapes of bacteria and their associated arrangements.

1.

ii. How are the subunits assembled into the cell wall?

1.

Biofilm antibiotic resistance

1. Slow Penetration * Because the films are so thick, it prevents the antibiotic to penetrate in the biofilm 2. Stress Response * Bacteria that are underneath the bacteria preventing the penetrate the antibiotic, start to respond the presence of antibiotics by producing different proteins that will actually allow to prevent and protect the bacteria from the antibiotic 3. Altered Microenvironment * The stress response bacteria produce send chemical signals that enable an altered microenvironment * This bacteria can produce different types of proteins that will protect them from the antibiotics 4. Persisters * Even if the antibiotic is able to kill most of the bacteria, we can see some persisters, so they will multiply again and repopulate the system.

Explain the relationships between organisms and their outcomes (e.g. parasitic etc)

1. Symbiotic Two organisms live together in a close partnership • Symbiotic Obligatory: When one or both members need the relationship to survive - if they get separated, they will die Three kinds: 1. Mutualism= (+) and (+) - when the relationships is obligatory and both members benefit Ex.: H. Sapiens (+), Bifidobacterium animalis (+) * Bifidobacterium animalis (+) it gets a food from the H. Sapiens, while the H. Sapiens benefit because Bifidobacterium animalis because it assists in our digestion of lactose. 2. Commensalism = (0) and (+) - when one organism benefits and the other is not harmed or aided Ex.: S. aureus (0), H. influenzae (+) * H. influenzae requires factor X as a growth factor in the media, but if we grow S. aureus on a blood agart plate with H. influenzae in the plate, the H. influenzae will thrive because the S. aureus while going about its normal processes actually processes the blood so the H. Influenzae can grow. 3. Parasitism = (-) and (+) - when one organism benefits (the parasite) at the expense of the other (the host) Ex.: H. sapiens (-), Ascaris lumbricoides (+) * Ascaris lumbricoides gets helped to a point that it kills its host. - The relationship types vary by whether or not each participant is unaffected (0), helped (+), or harmed (-) 4. Non-symbiotic - Free living organisms • relationships are not required for survival - non-obligatory A. Synergism - two or more organisms that cooperate to benefit all, but the interrelationships is not essential to survival Ex.: Cellumonas and Azobacter live in close association near plant roots and benefit from each other and the plant benefits by being infected because they fix nitrogen from the soil for the plant B. Antagonism - one member of the community of organisms acts to inhibit or destroy other members, but when they are separate they sthrive. Ex.: Clostridium difficile- causes colitis * Clostridium difficile it gets killed by our normal microbiota in our intestine

Describe the role of the macrophage, B-cell and T-cell (and related subtypes) in immunity.

27.

What is opsonization and why is it significant in the immune system?

29.

Define and cite examples of diffusion

3.

Explain the role of TLR in immunity.

30.

What are the signs of localized inflammation? How is it different from systemic inflammation?

31.

Describe what makes a "good" antigen.

32.

Recall the steps of endogenous and exogenous antigen presentation.

33.

Describe the function of a B-cell and T-cell receptor.

34.

Explain how T-cells are activated.

35.

Explain how CD8+ T-cells recognize and kill infected cells.

36.

Recall the types of CD4+ cells and the types of cells they go on to activate.

37.

Explain the parts of an antibody (e.g. hypervariable region, heavy chain etc) and the types (e.g. IgM, IgG etc).

38.

Describe how immunities are classified (e.g. natural passive, artificial active, etc)

39.

Recall types of hypersensitivities and what causes them

40.

Compare and contrast phenotypic versus morphologic classification schemes.

8.

Classify bacteria based on their carbon source (e.g. heteroptroph, autotroph, saprobe)

Carbon Nutrition - Microbes (and all living organisms) are placed in 2 groups depending on what source they use for obtaining carbon (in the form of carbohydrates). 1. Autotrophs - self-feeders - Use CO2 (inorganic, atmospheric) as a source of carbon for making sugars - They are: a. Photoautotrophs - Carbon source: Use SUN light for energy to make sugar from CO2 - have chloroplasts like plants Ex.: Cyanobacteria: Microcoleus chthonoplastes b. Chemoautotrophs - Carbon source: Use chemical energy in order to make their own carbs - Oxidize inorganic sources (sulfur, nitrogen) for source of energy to make sugar from CO2 - not medically important Ex.: Sulfur vent tube worms: Riftia pachyptila. They utilize sulfur that comes out of the ocean vents in order to make their own carbohydrates 2. Heterotrophs - other-feeders - use organic sources of carbon for food from other living organisms (dead or alive) - Can't make their own carbohydrates, so they need to consume already made carbohydrates a. Saprophytes - It is an Heterotroph - decomposers. * Most bacteria are considered decomposers - Carbon source: Gets its carbohydrates from dead organic matter (so they also live in dead organic mather) - Ex.: Monotrope uniflora: Indian Pipeb. Parasites i. Strict (Obligate) Saprophyte - an organism that lives exclusively on dead organic matter - cannot survive on living or in living organic matter Ex.: Maggot of Musca domestic b. Parasite - Heterotroph - Carbon source: get nutrients from a living host - lives inside of other living organism, usually in the other organism's detriment. Ex.: Giardia lamblia: Once an animal or person has been infected with giadia, the parasite lives in the intestine and is passed in the stool. i. Strict (obligate) parasite - an organism that can only grow in other living organisms - Must live inside another organism. Ex.: Chlamydia trachomatis is a human obligate parasite. It cannot survive outside a host and for this parasite, the host is always a human ii. - Is actually a SAPROPHYTE (that is, something that lives in dead organic matter) that infects a host (usually under conditions where the host is compromised) and becomes a PARASITE Ex.: Candida albicans is a kind of fungal infection. Survives well living on dead organic matter but if it comes in contact with a human can causes thrush in immuno-compromised patients. *Thrush is a parasitic infection of candida *. Chemolithotrophs

Taxonomy

King Phillip Came Over For Great Spaghetti (Kingdom, Philus, Class, Order, Family, Genus, Species) - Today, modern taxonomy reflect a PHYLOGENIC HIERARCHY 1. similar SPECIES are grouped into GENERA. 2. similar Genera is grouped into larger taxonomic categories: genera sharing common features are grouped together to form FAMILIES; 3. similar families are grouped into ORDERS; 4. orders are grouped into CLASSES; 5. classes into PHYLA, 6. and PHYLA into KINGDOMS.

Explain the role of normal flora in health and disease.

Normal flora - Also referred to as Normal Resident Microbiota or Indigenous Microflora. - For every cell in your body there are 10 microbial cells - You are not always sick because these microbes are known as normal resident flora. They are the bacteria that is always present in your body. - They live and thrive in a healthy host and do not cause disease under normal circumstances. However, occasionally they can cause disease. - These microbes have commensal (where the host will not be affected in any way) or mutualistic relationships (where both host and bacteria benefit from the relationship) with the human hosts. Colonization of flora -The female genital tract is colonized with the normal resident flora. These bacterias are very important because we are first inoculated with normal flora when you are born through the vaginal canal. - Colonization (n. 2 in the picture) begins at birth, with the normal flora present on the mother's vaginal canal that gets inhaled through the respiratory tract, swallowed and it also begins to colonize the skin. - Children born from C-sections that do not get exposed to the mother's normal flora will suffer from gastrointestinal issues, skin problems and some other issues. * Therefore, provided that the mother does not have any disease, the mother vaginal tract gets swabbed and then swabbing child's skin and mouth. - If the normal flora cross into sterile tissues, then you have an invasion (n. 3) - If the bacteria is able to invade, stablish itself and replicate, we call it infections (n.4) Microbiota • Microbiota present in our body colonize the outer surfaces of the body but do not penetrate the sterile tissue or fluids under normal circumstances. * If they do penetrate, then that is when they cause disease. • can either be: 1. RESIDENT: permanent part of the population of bacteria in our body. 2. TRANSIENT: Generally not permanent - they will be with us in a short amount of time before they are completely cleared from the body. Ex.: Over the county supplement of Lactobacillus or Acidophillus. Once you stop taking it, they will clear out of the body over a period of time. - Most of the normal flora is found in the Gastrointestinal Tract, but can also be found in Nasal/Oral Passage, Skin, Urinary Tract (male urinary tract is sterile), Genital Tract, and Upper Respiratory Tract (trachea, throat (pharynx), and voice box (larynx)) • Microorganisms that cause disease or have the ability to cause disease. • All pathogenic (disease causing) microbes have a PARASITIC RELATIONSHIP with the host * the parasitic bacteria is going to be helped but the host is going to be harmed. • Classified based on where they come from as: 1. OPPORTUNISTIC • Opportunistic pathogens are endogenous infections * Infections from the normal flora crossing to sterile tissues, where they do not belong 2. TRUE PATHOGENS • True pathogens are exogenous infections * they come from RESERVOIRS - external sources (either other people or surfaces) and they will make everybody they come in contact with sick. • Pathogenic microorganisms that infect a host can cause disease -Microbiota can be pathogens. * they will cause ENDOGENOUS INFECTIONS because they are OPPORTUNISTIC PARASITES - Microbiota become a pathogen if they are infecting: 1. UNUSUAL SITE • Ex. S. aureus is normally found in the skin where it does not cause disease. However, if it enters an Unusual Site, such as the eyes, it can cause conjunctivitis. In the skin, it can cause boils and carbuncles, in the bone can cause osteomyelitis, and if Ingested, it can cause Staph food poisoning. 2. IMMUNOCOMPROMISED • Ex.: Taking Steroids (which reduce inflamation and therefore suppress the immune system), have HIV/AIDS, is taking chemotherapy, or is too young or too old of age then your own microbiota can become pathogenic. 3. CHANGE IN OTHER MICROBIOTA IN OUR BODY • Ex.: Loss of antagonism (when one bacteria inhibits the growth of another) then the bacteria is allowed to grow and can make you sick. -> Diseases classification • In conjunction with the portal of exit and the mode of transmission, diseases are classified based on how they behave in the host and how the behave in a population 1. COMMUNICABLE DISEASE - from sick host to healthy host • Ex.: Chicken pox (Varicella zoster), typhoid fever (Salmonella typhi), TB (Mycobacterium tuberculosis) 2. CONTAGIOUS DISEASE - Type of communicable disease that spreads easily from host to host - a communicable disease that is spread easily from host to host • Ex.: Chicken pox (Varicella zoster), Flu (Influenza) 3. NON-COMMUNICABLE DISEASE - does not spread from host to host • Tetanus (Clostridium tetani), Botulism (Clostridium Botulinum), Lyme's Disease(Borellia burgdoferi) ** You could not get it from taking care of somebody with those illnesses 1. Function: • Normal flora prevent the overgrowth of pathogenic microorganisms • Normal flora are antagonistic of pathogen growth through competition 2. Example(s) a. Bifidobacteria are Gram-positive, non-sporeforming, lactic acid bacteria. Bifidobacterium bifidum is the predominant bacterial species in the intestine of breast-fed infants, where it antagnozies growth and prevents colonization by potential pathogens. These bacteria are sometimes used in the manufacture of yogurts and are frequently incorporated into probiotics. b. • Fusobacterium are anaerobic, non-spore forming, Gram negative bacteria • Colonizes oropharynx (causes gingivitis), intestine and colon (causes IBD and ulcerative colitis) • Although it can be part of the normal flora its presence is now considered pathogenic because it is linked to colorectal cancer * Fusobacteria genomes were found in 89% of the colon tumors sampled

Use terms such as bacteremia, septicemia, viremia etc, to describe what is happening in the blood during an

Sterile Tissues and Fluids • STERILE TISSUES include: heart muscles, nervous system, inner-most portion of the eye, blood * and the male urinary tract. • STERILE FLUIDS include: cerebrospinal fluid, aqueous humor of the eye, blood * Microbiota present in our body colonize the outer surfaces of the body but do not penetrate the sterile tissue or fluids under normal circumstances. * If they do penetrate, then that is when they cause disease. - If the bacteria remain in localized tissue, the infection is easy to be controlled. However, if the blood becomes infected, we usually have a more severe infection that is harder to clear up. - Blood is sterile, so when it becomes contaminated with microbes it develops into these states: (First stage) 1. Bacteremia- small numbers of bacteria in the blood 2. Viremia- a small number of viruses in the blood (not seeing through the light microscope - electron microscope would be necessary) 3. Fungemia- the presence of fungi, mold, yeasts in the blood (the second stage - develops into:) 4. Septicemia- a state in which microorganisms are rapidly multiplying in the blood and present in a large number * Not easy to recover from 5. Toxemia- a state in which a toxin is spread from the initial site of infection through the blood (Ex.: diphtheria, tetanus, botulism) - Bacteremia and septicemia are diagnosed by looking into blood smears and we can look at the number of bacteria present. * In a healthy person, no bacteria at all should be present.

Recall cells that make up the blood and their significance during different types of infection.

The blood contains both non-specific (innate) and specific defenses - Whole blood is liquid tissue that courses through the arteries, veins, and capillaries • Whole blood is made of blood cells suspended in liquid plasma • Serum is like plasma except that is separated from clotted blood - There are a number of blood cells present in a blood sample: 1. ERYTHROCYTES (Red Blood Cells) - Red blood cells (erythrocytes) - most abundant cell in whole blood - carry oxygen - do not have nucleus * all the immune cells have nucleus 2. PLATELETES (fragments of Megacarryocytes) - are not cells *they are fragments of the MEGACARRYOCYTE - required for blood clotting - Important function in inflammation 3. LEUKOCYTES (White Blood Cells) - INNATE immune System: - are cells of the INNATE and ADAPTIVE immune systems that fight infection by phagocytizing microbes. - Also kill infected cells, tumor cells, and recycle worn-out RBCs and dead tissue. - Include a. Neutrophills - Are phagocytic and motile. - Are very active in initial stages of infection * the number of neutrophils in a blood sample increase at the beginning of the infection - Diagnostic tool when we take a blood sample from a patient. * the profile of the number of neutrophils they have will determine what type of infection they have and how long the infection has been going on b. Basophiles - active in inflammation and in allergic responses - Responsible for releasing HISTAMINE c. Eosinocytes - Respond to EUKARYOTIC PATHOGENS. * Is gonna be present in large number when you have large eukaryotic pathogens. * It destroys large eukaryotic pathogens such as helminths and fungi d. Monocytes - MONOCYTES mature into MACROPHAGES - MACROPHAGES are the primary PHAGOCYTIC cell of the INNATE immune system. * are phagocytic, engulfing microbes - Disposes worn out red blood cells - ADAPTIVE immune system: • LYMPHOCYTES - Are the LYMPHOCYTES * Lymphocytes are also LEUKOCYTES (White Blood Cells). a. T-cells - Are the LYMPHOCYTE (adaptive immune system) kind of LEUKOCYTE (WBC) - function in cellular immunity b. B-cells - Are the LYMPHOCYTE (adaptive immune system) kind of LEUKOCYTE (WBC) - function in humoral immunity ---> they are the cells that MAKE the ANITBODIES

What is a fastidious microbe and how are its growth requirements different from other microbes?

What type of microbes would require a growth factor? - Fastidious microbes Ex.: Haemophilus Influenzae - Requires Factor X (which is a human clotting factor) --> Commensalist relationship: S. aureus (0), H. influenzae (+) * H. influenzae requires factor X as a growth factor in the media, but if we grow S. aureus on a blood agart plate with H. influenzae in the plate, the H. influenzae will thrive because the S. aureus while going about its normal processes actually processes the the blood so the H. Influenzae can grow.

Cell Wall

c.