MIDTERM 1 MICROBIOLOGY COMBINED
Ribosome
○ Site of protein synthesis ○ 70S made up of 30S and 50S subunits ○ This difference from eukaryotic cells (80S made up of 60S and 40S) allow for the use of antibiotics that only target bacterial ribosome
Microorganisms are Everywhere
- food - environment - on and in the human body (90% of all cells are bacterial, fungal or otherwise non-human)
Agar Concentrations in media
0.05-0.1% Liquid Slows down mixing of fluid Reduce convection currents Thioglycollate broth 0.2-0.5% Semi-solid Determine motility Prevents dispersion of acid/alkaline products Motility media O/F sugars 1-2% Solid Solidification MacConkey Blood Agar 2-5% Solid Prevent swarming growth MacConkey (may have even more, proteus is very mobile)
Culture
: Organisms that grow and multiply in/on culture media are referred to as a culture
Criteria Media Must Meet
In order to grow microorganisms media must: Contain the right nutrients Oxygen or other gases must be available if required Have sufficient moisture Proper PH Be sterile Most important criteria, want to ensure that bacteria is coming from the specimen and not from something else. Be incubated at proper temperature and atmosphere
DensiCHEK Plus
A photometer which provides values in McFarland units, proportional to microorganism concentrations. It will tell you what McFarland standard you have, aiming for somewhere between .5 and .63. Measures the absorbance of particular wavelengths (580nm) of light by a solution. The concentration of the substance being measured is proportional to the intensity of the color of the solution. The darker the color or more turbid the liquid, the higher the McFarland reading. Must be zeroed before use (blank) Must be calibrate using standards.
Agar with peptic Digests of Blood
Add the enzyme (pepsin) under acidic conditions and increased temperature to whole blood Contains a number of growth factors, coenzymes and hemin Added to agar for recovery of specific organisms Example Filde's agar for recovery of Haemophilus sp. (don't need to know)
polymerase chain reaction
Advent of PCR initiated a revolution in microbiology - Molecular diagnostics Simple, sensitive and powerful A single copy of target DNA can be amplified into an exponential amount of nucleic acid product in 25- 40 reaction cycles
Mixed Acid vs 2,3 Butanediol
After an organism metabolizes glucose to pyruvic acid in glycolysis, there are many pathways it can follow. Mixed acid fermenters are further divided into two groups: Those that produce mixed acids with a highly acidic pH end product but no 2,3 butanediol Those that produce 2,3 butanediol with a neutral pH end product acetoin This is important when trying to understand the MR/VP test
Peptones and Hydroylsates
All media will require a protein source (nitrogen is needed for building) If a bacteria uses any type of protein it will create an alkaline environment in the media, thus a media might have a pH indicator. If you see alkaline, that is what is happening. If a bacteria ferments a carbohydrate it will create an acid. Proteins are essential to the structure and function of bacteria A source of nitrogen Are water soluble Relatively heat stable- withstand autoclaving Peptones are made from proteins by hydrolysis or by enzyme digestion A lot of bacteria can't make use of the protein when it is in its fully folded form, thus they often add it in the form of a polypeptide or a peptone, so the bacteria can access it. Enzyme breaks down meat or milk to amino acids, peptides or poly peptides Tryptone best choice in bacterial media since used by most bacteria It is made by breaking down milk protein (casein) using the enzyme trypsin
Peptidoglycan
Also known as murein Composed of sugars or polysaccharide chains and peptides or amino acids Sugar portion are alternating N-acetyl-d-glucosamine (NAG) N-acetyl-d-muramic acid (NAM) Attached to the NAM is a peptide chain of 4-5 amino acids L-alanine, D- glutamine, L-lysine, D-alanine These peptide chains can be cross-linked to the peptide chain of another strand with a pentaglycine bridge forming a 3D mesh Think of a basket weave pattern joined by a pentaglycine bridge Back bone of the cell wall.
Carbohydrate Concentration in media
At 0.5-2% the acid created can be used to determine if it is a fermenting sugar.
VITEK 2
Automated system originally designed for the space program Identifies bacteria (aerobic or facultative gram pos & neg) and yeasts Non fastidious bacteria Can't ID TB or fungus Cards have 64 wells that each contain a test substrate Measure metabolic activities such as acidification, alkalization, enzyme production, and growth in the presence of inhibitory substances Uses a photometric system to detect changes in light like turbidity or the colored end products of substrate metabolism Each test reaction is read every 15 minutes Results from 4-6 hrs. (some bacteria longer) Performs susceptibility testing of the non-fastidious bacteria
Blood and blood Products
Blood is added to base media like Trypticase soy agar or Columbia base agar Used to enrich media so more fastidious organisms can grow Most bacteria grow healthier Cannot be autoclaved therefore blood added to agar after sterilization It has to be added after Therefore must be collected from animal by aseptic technique Horse or sheep And media checked for sterility before use Blood used must be anticoagulated or defibrinated
Crystal Violet - Primary Stain
Blue purple in colour, thus when you first stain them... Everything will be all purple whether or not they are gram negative or gram positive. Basic stain Positively charged stain (will attach to negatively charged items) Diffuses through cell wall pores & bonds with negatively charged cell groups (e.g. phosphate groups in nucleic acid)
C&S
C & S (Culture & sensitivity testing): term used to describe the test for culturing bacteria from patient specimen in hospital lab Means that the doctor wants you to culture it and perform susceptibility testing.
Inoculum
Introducing microbes into/onto culture media to initiate growth
TSI Results Intepretation
CO2 or H2 Gas Detection: Positive Gas production is indicated by the following: Gas bubbles will be seen throughout the medium Cracks in the agar also is due to gas production Lifting of the agar Gas production can be seen in any of the following areas on the TSI medium: Butt (usually) Stab line Sides H2S detection: Na thiosulphate in TSI media provides sulfur Some organisms can liberate the sulfur from this compound and form hydrogen sulfide The newly formed H2S (hydrogen sulfide) reacts with ferrous sulfate to yield ferrous sulfide which is an insoluble black precipitate Blackening of the medium is almost always observed *********** (bottom) of the medium H2S + FeSO4+ → FeS + H2SO4
Microscopic Examination
Can be done directly from the specimen (swab, fluid, tissue...)--> can quantitate # of bacteria From a colony growing on media --> do not quantitate
Core
Commonly contains carbohydrates, phosphates and amino acids
identification and susceptibility Results
Computer software will analyze data Report (identification or susceptibility report) will be generated Aiming for 85% or higher ID Even if the VITEK calls something a 99% match, you always check your purity plate to confirm its characteristics Report can be printed or sent to LIS Purity plates Always make a purity plate anytime you make a suspension of the organism and are running it through automation or API. Want to ensure that a clean sample was run through your testing system.
Cycles of PCR
Cycles: each made up of 3 steps Denaturization of target DNA using heat (95-98C) Sequence specific primer anneals to the target sequence (40-60C) Primer extension by Taq polymerase at 72C
Enzymes in DNA Replication
DNA helicase: separates ds DNA to ss stranded DNA DNA polymerase: adds nucleotides one by one to make a new strand complementary to the original DNA is synthesized in the 5' --> 3" direction However polymerase can only add nucleotides to the 3' end of DNA A primer which is a short stretch of RNA (5-10 nucleotides long) must be added Primase enzyme makes an RNA primer that provides the 3' end for the polymerase to work on - therefore new DNA strand is built in the 5' --> 3' direction
Quality Control for VITEK
Daily Temperature Back up Empty discard bin Weekly: cleaning Monthly: Optics Densicheck: Battery check & calibrated daily
Determine how many organisms are responsible for an infection
Determine whether infections caused by a single organism = monomicrobial Or by multiple organisms = polymicrobial
Gram Stain
Devised by Hans Christian Gram 1884 Simple, most commonly used stain in Microbiology Used to differentiate bacterial species into two large groups - gram positive and gram negative
Identification Test Tube
Dispense 3mL 0.45% saline into plastic vitek tube Have to dilute the McFarland tube Inoculate organism using stick or or swab (ID suspension) Use DensiCHEK: 0.5-0.63 density
Susceptibility Test Tube
Dispense a second tube of saline (3mL) Have to dilute the McFarland tube Take 145µL (GN) OR 280µL (GP) from ID suspension - Pipettors are color-coded Transfer the proper amount into that second tube with pre-dispensed saline
Card Reading
Each card moves into place - read every 15 min
Quick Review of DNA
Each strand of DNA is a large molecule made up of many repeated subunits -nucleotides Each nucleotide is made up of a five-carbon sugar (deoxyribose) a phosphate group and a nitrogenous base One nitrogenous base -either a purine or pyrimidine is in each nucleotide subunit The purine bases are adenine (A), and guanine (G) The pyrimidine bases are cytosine (C) and thymine (T) Guanine must always pair with cytosine Adenine must always bond with thymine These are called "base pairs" & form bridges across the backbone structure of the DNA strand (like rungs on a ladder)
Iodine (Mordant)
Everything is still all purple. Diffuse into the cell Combines with the already bound crystal violet forming crystal violet-Iodine (CVI) complex This is a larger dye complex Traps crystal violet making its exit from the cell difficult CVI complex is stable in water but soluble to solvents such as alcohol & acetone Will come out if you add these.
Extracts and Infusions
Extract Made by slow cooking a protein source until concentrated to a paste Broken down the protein to its smaller form, but also has all the other nutrients Infusion Made by leaving protein source in water for long periods of time until liquid infused with protein Allow it to sit in a cold pot of water and allow all the vitamins and smaller forms of protein to come out of it Both provide an extra source of vitamins and organic growth factors missing in common peptone formulations Are heat stable - can add to media before autoclaving There are meat or yeast extracts available Common infusions made from calf brain or beef heart This allows you to breakdown the whole organ, and also allow you to get additional nutrients Method use depends on what the end goal is.
Lipopolysaccharide Layer
Found in gram negative bacteria only Made up of three basic parts Layers consist of O antigen (very antigenic; many antibodies made against this), core polysaccharide, and Lipid A
TSI test principle
Glucose 0.1% If an organism can ferment glucose (a monosaccharide) it will be used first Acid is produced; will initially turn the entire medium yellow (slant & butt) Low concentration of glucose is depleted rapidly Organism now breaks down the peptones in the presence of O2 on the slant & produces ammonia Leads to a reversion to alkaline pH in the slant =orange slant Butt is ANO2 so stays acidic -more acid produced in fermentation Final result if the organism uses glucose only: Alkaline slant orange and acid butt yellow Lactose 1.0% & Sucrose 1.0% If the organism is able to use glucose and one or both of the other two sugars, there is a lot of acid formed Due to the large concentration of these carbohydrates in the media they are not depleted in 18-24h There is no need for the organism to utilize the peptones and no reversion occurs Final result if the organism uses glucose & lactose or sucrose or all three: Acid slant and butt both remain yellow
Gram positive vs gram negative cell wall
Gram positive cell wall: Thick, peptidoglycan layer (90%) Many teichoic acid cross-linkages Lower lipid content , 0-2% No periplasmic space Resists decolourization The stain wont be removed. Gram negative cell wall: Thin peptidoglycan layer (5-20%) No teichoic acid cross-linkages Higher lipid content 10-20% Have both an outer and inner membrane Has periplasmic space Does not resist decolourization The stain will be removed.
Carbohydrates in media
Has a protein source, pH indicator & 1% of the carbohydrate being tested Fermenters: Produce lots of acid so indicators are used which change colors below pH6.0 like Phenol Red Oxidizers: Produce less acid so indicators are used for weak acids and bases like Bromothymol blue If glucose and lactose present, bacteria will use glucose first and lactose next Certain bacteria use glucose only - Bacteria incapable of fermenting glucose can't use lactose
Media with Added Serum
Horse serum most often used Enriches and helps solidify media Helps coagulate the agar, and adds different nutrients that can only be found in serum. Produced through Inspissation Inspissation = Heating of agar & serum to 850 C so the proteins coagulate and solidify the media Example: Loeffler's medium for C. Diphtheriae (don't need to know)
Barcode Reader
Identifies cassette & each card (type, expiry) "Happy sound" when it matches the virtual cassette you created
Lactose Fermentation
Lactose fermentation: lactose --> glucose + galactose In order to ferment lactose bacteria must produce: β-galactoside permease to allow lactose to enter the bacterial cell wall And inside cell --> B-galactosidase to break down lactose Bacteria with both enzymes = LF Bacteria with permease but no β-galactosidase = NLF Bacteria that possess β-galactosidase but no β-galactoside permease may mutate and, over a few days or weeks, begin to produce the permease enzyme= LLF (late lactose fermenters)
Agar with Laked Blood
Less RBC, cellular components destroyed than with heating --> style lyses it but not just by heating it, you want to maintain components that are ruined by lysing it. Procedures used make Laked blood: Repeatedly freezing & thawing whole blood so heat-sensitive ingredients not lost Adding a lysing enzyme like saponin to whole blood Adding an equal volume sterile distilled water to whole blood Examples of media with Laked blood: (don't need to know this for tests) Hoyle's medium (Corynebacterium diphtheriae) Kanamycin Vancomycin Laked Blood agar(KVLB)
Lipid A
Lipid A is the endotoxin produced by Gram-negative bacteria Responsible for activating complement, inducing secretion of tumor necrosis factor, and interleukin causing fever, diarrhea, and possible fatal endotoxic shock Can't release out into the environment, the only way it can be released is if that bacteria gets killed. Not specific to any cells, causes a general immune response (fever, rash, inflammatory)
O/F Considerations
Loosen caps to permit air exchange so that organisms that are neither O or F can utilize proteins and create alkaline conditions Do not use mineral oil because it increases air diffusion Slow growers may require 3-14 days to show a result -ideally, any that are negative for O or F at 48h should be incubated longer O/F media can also be used to determine motility because media is a semi solid Do not use Staphylococcus from a mannitol salt plate
History of Culture Media
Louis Pasteur first used simple broths - urine or meat extracts Only detected growth (turbidity) or no growth (no turbidity) Doesn't allow you to determine any morphology or even if the bacteria was still alive. Robert Koch realized the need for solid media - used potato pieces to grow bacteria Tried to use gelatin as solidifying agent but it liquefied at 35 C & could be digested by bacterial enzyme gelatinase Agar discovered as a good way to solidify culture media Petri dish developed in 1887
Why Culture Media Became Necessary
Microorganisms seen for the first time under the microscope Controversy began over whether bacterial growth was spontaneous Louis Pasteur disproved this using a Pasteur flask A method for growing bacteria (culturing) was needed in order for this belief to be refuted And to advance the science of Microbiology
AUTOMATED TESTING
Miniaturized testing for identification and susceptibility results Freeze-dried substrates Detect preformed enzymes Color change: Acid or alkaline pH changes Chromogenic -colorless to start if enzyme present becomes colored Fluorogenic -non-fluorescent to start, if enzyme present they fluoresce Turbidity: increased cloudiness if bacteria grows Increased accuracy and faster results, 4 to 6 hrs.
Acetone Alcohol (Decolorizer)
Most important step .... Timing is critical Composed of equal volumes of 95% ethanol and acetone Extracts lipid creating larger pores in cell wall; cell wall more permeable Dehydrates peptidoglycan reducing pore size & movement In GN cell wall: Has high lipid, low peptidoglycan Thin peptidoglycan isn't enough to hold the CVI complex. Lipids extracted/lost and CVI complex leaves the cell In GP cell wall: Has low lipid, high peptidoglycan Peptidoglycan shrinks and CVI complex trapped in the cell Looking under the microscope at this point: GN will look colorless and GP will appear bluish purple
Culture Medium
Nutrient material for growth of microorganisms in the lab
ONPG Method
ONPG tube media or the ONPG disc are both commercially available - in MIML113 we use the disc Take a small glass test tube & add 0.5ml of saline Using a straight wire, inoculate heavily (1-2 colonies) into the saline Add an ONPG disc and para film the tube Incubate overnight in O2 at 350 C Any shade of yellow is a positive result
Automated Detection Systems
Often have a light reader at the end of the machine, which will read light transmittance at the beginning and at the end after incubation to determine if there has been a chance Photometry- device to measure the intensity of light through turbidity or color change Spectrophotometry- a type of photometer that detects changes in light by the ability of a substance to absorb or transmit light Looks at the wavelength of light Fluorometry- detects changes in light through increasing fluorescence After incubation (some systems require reagent addition), detector reads & interprets the tests -numeric biocodes with computerized databases -often interfaced with the LIS
Annealing
Oligonucleotide primers are annealed to the denatured ssDNA strands One to bottom strand & one to the top strand
O Antigen
Outermost layer Highly antigenic O-specific polysaccharide Allows for serotyping of some gram negative organisms to aid in identification
Triper Sugar Iron
PURPOSE: Detects the fermentation of 1) glucose, lactose, and/or sucrose, 2) gas production (H2, CO2), 3) H2S production Used in the presumptive identification of many Gram negative bacilli (Enterobacteriaceae); useful in screening intestinal (enteric) pathogens INGREDIENTS Oxoid media: Peptone Glucose 0.1%, lactose 1.0%, sucrose 1.0% pH indicators: Phenol red H2S indicators: Ferric ammonium sulfate (or Ferric ammonium citrate) and sodium thiosulfate
Oxidation Fermentation Test (O/F)
PURPOSE: Determines if organism is capable of metabolizing CHO aerobically (oxidative) anaerobically (fermentative)or not at all To differentiate non-fermenting strict aerobic GNB such as Pseudomonas from Enterobacteriaceae INGREDIENTS: Glucose at 1% reduced Peptone content, Bromothymol blue indicator Semi-solid agar Un-inoculated media is green
Methyl Red Test
PURPOSE: Tests the ability of an organism to produce stable mixed acid end products from glucose fermentation and to overcome the buffering capacity of the media Useful in the identification and differentiation of Enterobacteriaceae MAIN INGREDIENTS: Peptone High concentration of buffers Glucose Methyl red indicator added after incubation
TSI Method
Pick a single pure colony using the sterilized wire. Streak the surface of the slant in a "zigzag" fashion Stab down the centre of the medium to within 2 mm of the bottom(butt) Loosen the caps of the tubes prior to incubation Incubate the tube for 18 to 24 hours at 350C in O2 incubator Read reactions no earlier or later than 18 to 24 hours of incubation
Safranin Counterstain
Positively charged, will now attached to any negatively charged areas that don't have a stain (all the GP that are now colourless) Can also use dilute Carbolfuchsin No effect on GP as binding sites were blocked by crystal violet (remember CVI complex) Forms bonds with cell groups in GN cell wall as binding sites are now exposed Looking under the microscope at this point: GP are bluish purple and GN are pink
Gram Stain from a Colony
Preparation of Slides: Use a sterile glass slide with a frosted end Label the slide with specimen # or organism name, MB#, & date Place one small drop of sterile saline in the center of the slide Touch one colony that you wish to stain using a loop, swab or wood stick Don't pick up too much colony, otherwise you won't be able to make any differentiations. (barely touch) Mix the bacteria and saline so it forms a 2cm circle (Don't make it too big) Want to create a "monolayer" (think of heme) This will allow you to determine shape and arrangement. Air dry - do not place on slide warmer until dry If you heat fix it while it is still wet, you will not get the right shape and arrangement. "Fix" material to the slide using heat or methanol
Agar with Whole Blood
Provides wide range of essential nutrients 5- 10 %sheep or horse blood commonly used Human blood not recommended - may contain antibiotics or antibodies inhibitory to bacteria Examples of media with whole blood = Blood agar & CNA agar
O-NITROPHENYL-β-D-GALACTOPYRANOSIDE (ONPG)
Purpose: To differentiate LLF from NLF - detects permease negative, β galactosidase positive organisms Biochemistry Involved: ONPG is similar to lactose except the glucose has been replaced with o-nitro phenyl ONPG is small enough to passively enter the bacterial cell wall without the permease enzyme Bacteria with the enzyme β galactosidase will degrade ONPG (colourless) to ortho nitro phenol (yellow) and galactose.
TSI Result Interpretation
Reaction TSI result Explanation Acid/Acid Yellow slant/Yellow butt Glucose and lactose and/or sucrose fermented Alkaline/Alkaline Red slant/Red butt No CHO fermented; aerobic organism Alkaline/Acid Red slant/yellow butt Only glucose fermented
Agar with Lysed Blood
Red blood cells broken down by heating (85 C) Cellular components released - nutrients more readily available for bacteria Begin the breakdown process for bacteria, and this makes it easier for the bacteria But some ingredients destroyed (e.g. enzymes) Examples of media with lysed blood New York City agar (w antibiotics) Chocolate Agar -2% hemoglobin - growth supplements Dark colour due to lysing Hemoglobin can be made by washing & autoclaving whole blood Choc used especially for isolation of certain fastidious organisms (example Haemophilus influenzae) Most organisms will grow on it, but it will grow even the fastidious ones.
Sealer Unit
Seals each card by bringing a heated wire across the transfer tube, cutting and sealing simultaneously Remaining plastic end drops into the tube Each card is moved into the carousel for reading
use the stains to help presumptively identify bacteria can show
Shape Arrangement Being able to read the gram stain will allow you to make deductions.De
MR Principle
Some bacteria ferment sugars by the mixed acid pathway Mixed acid fermentation results in highly acidic end-products, mainly lactic and acetic acid Cause a very low pH (< 4.2) in the medium which overcome the buffering capacity of the broth Results in a red color with methyl red indicator Other organisms ferment sugars via the butanediol fermentation pathway Produce smaller amounts of less stable acids & cannot overcome the buffering capacity of the broth Methyl red indicator turns yellow
Carbohydrates
Source of energy & cellular building blocks (e.g. carbon) Used also to detect fermentation reactions (with added indicator) Monosaccharide Glucose Disaccharide Two saccharides Lactose (glucose and galactose) Polysaccharide Starch
Agar
Source: red purple marine algae Complex water soluble polysaccharide Good transparency Resists digestion by bacterial enzymes Inert: Little effect on bacterial growth Bacteria won create bacteria against it Solidifying agent Melts easily at ≥ 85C - Solidifies at 30- 40C Remains liquid at 45C or > so able to pour media at optimum cooled temp of 55-600C Allows you to add ingredients that would otherwise be ruined by heat. Withstands the rigors of streaking
What determines staining
Staining results are based on cell wall structural differences of gram positive and gram negative organisms Based on cell wall differences Allows us to observe various shapes and morphological arrangements of bacteria Used as the first step in presumptive identification - help guide physicians to early treatment
Transmittance Optics
Starts with a baseline Monitors growth and activity in each well LEDs produce light Measures growth by how much light is prevented from going through the well by increased turbidity or changes in color intensity
Not all specimens have a direct stain
Stool sample: looking for food poising... You will have alot of different organisms and a lot of different species, this wouldn't tell you from a gram stain as it would be impossible to read.
O/FTest Method
Take 2 tubes for each organism you are testing One to test in aerobic conditions -open tube One to test in anaerobic conditions-closed tube Pick a single colony of bacteria from 18-24 hour culture Use a straight wire to stab down into the center of the media to within ¼ inch from the bottom Do the same in the second tube Pour 1 cm of sterile mineral oil over one inoculated tube = anaerobic conditions (Closed tube) Leave the other inoculated tube without sterile mineral oil = aerobic conditions (Open tube) Incubate at 35o C for 48h or longer (due to time constraints we will only incubate for 24h here at Michener)
Denaturation
Target DNA double strand is separated into single strands
TSI Considerations
Test invalid if the butt of the agar is not stabbed or a pure colony is not used Don't use a loop to inoculate the TSI or it will look like a crack in the media which is falsely positive for gas If air is excluded (caps too tight), slant will not revert to red = a false acid result (leave caps loose) Ferrous sulfate (in TSI) is a less sensitive indicator of H2S than other ferrous salts - may cause discrepancy between TSI & SIM If read before 18h a false positive of A/A may occur If read after 24h a false NA/NA may occur due to all the sugars being used up and only peptones being left to use If H2S is obscuring the butt result assume the reaction *********** is acid
carbohydrate utilization
Tests that divide groups of bacteria by their ability to use specific carbohydrates And which metabolic pathway they use to break these carbohydrates down Breakdown can be achieved: Anaerobically through fermentation -high acid end products Aerobically through oxidation- Much less acid produced Asaccharolytic - don't use CHO; use other organic molecules for energy and carbon sources- No acid
Teichoic Acids
Think of them as the steel beams that give a high rise building support Polysaccharides present only in Gram positive (GP) cell wall Lipoteichoic acids are anchored to the plasma membrane and extend into peptidoglycan layer to surface Wall teichoic acids are attached to peptidoglycan and extend out to surface Can act as antigenic determinant & thus, used in serological identification Can promote an inflammatory response -Involved in adherence of bacteria to host cell Because they both stick out, they are antigenic. Provides extra rigidity to the cell wall
VITEK 2 Compact Cards
Warm card to room temperature GNI for gram neg Id or GPI for gram pos Id GNS for gram neg AST or GPS for gram pos AST Place card in cassette Each cassette holds 10 cards Each cassette has a barcode and a number on the front Place corresponding tubes in stand beside them Enter cassette" on computer & place in filling unit
Cell Wall
○ Provides shape, structure support, and osmotic protection ○ Composition depends on microorganism type § Peptidoglycan (murein) and lipid (most bacteria) § Chitin (fungus) § Mycolic acid (acid fast bacteria) □ TB § Mycoplasmas have no cell wall at all □ 99% of bacteria DO have a cell wall however. Confers gram staining characteristics of bacteria - *antigenic*
Basic Bacterial Growth Requirements
Water Distilled or deionized water used because free of impurities Amino-Nitrogen source Peptones, meat infusions or meat extracts Proteins Energy source Sugars, carbohydrates Allows ATP to be made Essential elements Basic building blocks of cells- carbon, nitrogen, sulfur, calcium, magnesium, potassium, iron, phosphorus) Trace elements - metal ions required by some bacteria Sodium, zinc, molybdenum, cobalt, copper, etc.. Used in enzymatic reactions Organic growth factors Blood, serum, yeast extract, vitamins, NAD
Bacterial Genetics Chromosomal
• Bacterial genes are found on a chromosome free floating in the cytoplasm • A single, closed, circular piece of double-stranded DNA • Supercoiled to fit the cell • Encoded with directions for :protein synthesis, cell growth, replication and survival
Clinical Microbiology
• Clinical microbiology ○ Investigation of human infection by pathogens such as bacteria, viruses, fungi and protozoa ○ Includes epidemiology, infection control, transmission, prevention, and treatment.
Bacterial Replication
• Duplication of chromosomal DNA for insertion into a daughter cell • Begins at a specific positions in the chromosome called "site of origin" where initiator proteins bind to DNA to start the process • At origin point, two replication forks form and enzymes move in opposite directions to reform two identical double strands of DNA, one for the original, and one for the daughter cell • This is called binary fission. This will produce an identical daughter cell
What is Microbiology?
• Microbiology is the study of very small living organisms or "microbes" • The microbial world includes: ○ Virus ○ Bacteria ○ Fungus ○ Parasites
Genetic Mutations
• Mutations : changes in the original nucleotide sequence of an organism genotype ○ Spontaneous ○ Due to an error during replication ○ Chemical or physical factors in the environment ○ Introduction of foreign DNA into the cell by mutagens such as plasmids, transposons, or bacteriophages § May kill the cell § Not be detectible §Provide a survival advantage like toxin production or resistance to antibiotics.
Mutagen - Plasmids
• Smaller circular double stranded DNA outside the chromosome but still in the cytoplasm - not essential for cell survival ○ Bacteria can have 1-100s of plasmids ○ Not apart of the circular chromosome. • Can replicate independently of chromosome through conjugation-horizontal transfer from one bacterial species to another ○ This will cause the bacteria to have unique DNA • Or replicate with the chromosome and be passed on to a daughter cell • Contain genes encoding for antibiotic resistance, toxin production virulence factors, and proteins for transfer of plasmid to other bacteria. Does not contain any genes that encode for the survival of the cell.
Constitutive and Inducible Genes
• Some genes are always expressed = constitutive • Some genes are silent and are expressed only under certain conditions = inducible
Bacterial Shapes and Arrangements
• Three basic shapes ○ Cocci ○ Rods ○Spirochete
Chromosome
○ A single negatively charged supercoiled double strand of DNA ○Small amounts of RNA, RNA polymerase and other proteins
Endospore
○ A survival response to adverse conditions in the environment § Thickening of the cell wall § "hibernation mode" ○ Resistant dehydration, chemicals and temperature change ○ Germinate under favorable nutritional conditions § Activation process that involves damage to spore coat Spores are not reproductive structures
Cytoplasm
○ Amorphous fluid with carbohydrates, proteins, enzymes, other metabolites and ribosomes
How does the capsule affect gram staining and morphology?
○ Bacteria will appear like snot (macroscopically) ○ Gram stain § The organism will stain, but you can see a clearing around it (this is the capsule)
What is the Capsule Made of?
○ Made of polysaccharides
Pili or Fimbriae
○ Not all bacteria make these ○ Tubes of protein (shorter than flagella) ○ Virulence factor - allow bacteria to adhere or colonize to surfaces § Proteins that match receptor sites in your body so that they can match to that cell and infect it ○ Assist in transfer of genetic material between cells in conjugation (promote attachment to other cells) Have nothing to do with motility - *antigenic*
Capsule - Main Functions
○ Not all bacteria posses the ability to make this ○ Main functions § Adhesion: allows it to stick to things § Survival : the proteins on the outermost of the cell wall are often recognized by the immune cells, if a bacteria has a capsule on it, it can hide the proteins from the immune cells, and can evade the immune system. ○ Protects bacteria from immune system antibodies and phagocytosis ○ Barrier against some antibiotics ○ *Antigenic* The immune system can recognize the proteins in the capsule, and can illicit and immune response.
Plasma, Cytoplasmic or Cell Membrane
○ Phospholipid bi-layer ○ Contains enzymes involved in ATP and phospholipid synthesis as well DNA replication ○ Selective permeability and active transport of material in and out of the cell § Semipermeable Site where metabolism occurs
Flagella
○ Protein appendages for motility § No other purpose ○ Highly antigenic ○ Not all bacteria posses this ○ Different kinds § A = polar flagella § B= polar lopotrichous flagella § C= amphitrichous flagella § D= Peritrichous