Incomplete Materials in the Final Lab Exam (Nov. 28/29): Labs #9-23 (All labs after the main lab exam) Please likewise read the survey sheets I gave out on Nov. 20 in lab. I will have office hour M/Th: 9:15 a.m. - 10:15 a.m. at DH 553 or DH 543 (my lab)Slide 2
LAB 9: Single Colony Isolation Know how to acquire single settlements through the "Streak for Isolation" on an agar plate method.Slide 3
Streak Plate methodSlide 5
Lab #9 Micrococcus luteus Staphylococcus aureus All in NA plates Serratia marcescens Klebsiella roseaSlide 6
Blood agar (BA) is a differential medium. - Some microbes create a chemical that can lyse RBCs – this process is hemolysis . - By developing microbes on blood agar we can figure out whether the bacteria deliver hemolysin and in this manner lyse the RBCs. - Blood agar is NA to which sheep RBCs have been included. - If hemolysin is created by the microorganisms it will be emitted into the medium and the RBCs will be lysed (the medium will be clear instead of red). - So nearness of clearing around the bacterial development shows hemolysis. - Growth on BA separates between the hemolytic and non-hemolytic microscopic organisms.Slide 7
Gamma hemolysis = No hemolysis Alpha hemolysis = Partial Beta hemolysis = CompleteSlide 9
Lab 10: Stock and Working Slants Why did we set up a stock and a working stock inclination for the obscure? Why did we develop the obscure in various media and under various conditions?Slide 10
"Working" "Stock"Slide 11
Lab 11: Simple Staining & Bacterial Smear Understand straightforward, negative, and positive recoloring. Know how to set up a microscopic organisms spreadSlide 12
Demos : straightforward stains of: Neisseria (diplococci) Pseudomonas (bacilli)Slide 13
Lab 12: Differential Staining (Gram Stain) Know the whole Gram Staining methodology and the capacity of every progression. Know the Endospore technique (in Appendix, p. 121)Slide 14
GRAM STAIN E coli (Gm - ) Staph epidermidis (Gm+)Slide 15
ENDOSPORE STAIN Bacillus megaterium See Appendix IV, p. 121 Outcome for endospore + for Micr20 Bacillus anthracis Clostridium tetaniSlide 16
Cell Arrangements:Slide 17
Lab 13: Selective and Differential Media EMB: Eosin-Methylene Blue a. Differential and specific properties. b. Contains bile salts and the colors eosin and methylene blue; all inhibitory to Gram-positive microbes (e.g. Staphylococcus aureus ). c. Selects for Gram-negative microbes (e.g. Escherichia. coli ). d. Separates lactose maturing ( dull shading with metallic sheen ) from non-lactose-aging ( dry ) microorganisms.Slide 18
Salmonella pullorum E. coli Staph. epidermidis Staph. aureusSlide 19
Lab 13: Selective and Differential Media TGA: Tellurite Glycine Agar a. Chooses for coagulase-positive staphylococci. b. Differential : coagulase-positive cocci shape dark states. c. Coagulase-negative cocci are for the most part repressed. The ones that develop are dark. d. Most Bacilli and Pseudomonas (Gm+) are restrained. e. Proteus sp once in a while develops and frame chestnut provinces.Slide 20
Staph. aureus E. coli Staph. epidermidis Salmonella pullorumSlide 21
Know every one of the media we canvassed in Micro20 since lab #9: The motivation behind the medium, how to peruse a positive and a negative outcome, what those outcomes mean, and the MAJOR parts of the medium.Slide 22
Lab 14: Antibiotic Sensitivity Antibiotics are chemicals that are delivered by other microbes/parasites that can avoid different living beings (microscopic organisms) from developing or slaughtering them. Affectability X Resistance to anti-infection agents. Bacteriostasis (halting bacterial development) X bacteriocide (executing of microorganisms). Expansive range anti-infection agents - powerful against an extensive variety of microorganisms (G+ and G-). Contract range anti-infection agents - viable against a little particular gathering of microscopic organisms (either G+ or G-).Slide 23
Lab 14: Antibiotic Sensitivity; Disk Diffusion MethodSlide 24
LAB 15: Catalase, Amylase, Gelatinase (Proteinase), MRVP, see Appendix IV, p.118-119Slide 25
CATALASE H 2 O 2 Negative Positive Enterococcus faecalis Streptococcus aureusSlide 26
Amylase: Starch Hydrolysis BEFORE AFTER Flood with Iodine arrangement E.coli Bacillus subtilis E coli - (neg.) Bacillus subtilis + (pos.)Slide 27
Gelatinase test: Plate was overflowed with Frazier\'s Developer Gelatinase + Gelatinase negativeSlide 28
LAB 16: Urease, SIM agar, CitrateSlide 29
UREASE TEST: Urease is a compound that breaks the carbon-nitrogen obligation of amides (e.g. urea) to frame carbon dioxide, smelling salts, and water. Individuals from family Proteus are known to create urease. At the point when urea is separated, alkali is discharged and the pH of the medium increments (turns out to be more essential). This pH change is recognized by a pH pointer that turns pink in a fundamental domain. A pink medium shows a positive test for urease.Slide 30
SIM agar SIM = S ulfide, I ndole, M otility. INDOLE TEST : Indole is a part of the amino corrosive tryptophan. A few microscopic organisms can separate tryptophan for nutritious needs utilizing the protein tryptophanase . At the point when tryptophan is separated, the nearness of indole can be recognized using Kovacs\' reagent. Kovac\'s reagent , which is yellow, responds with indole and produces a red shading on the surface of the test tube.Slide 31
SIM agarSlide 32
MOTILITY Motile bacterium Non-motile bacterium (e.g. Staph aureus ) (e.g. Pseudomonas aeruginosa )Slide 33
Citrate: The Simmon\'s Citrate medium tests the capacity of the bacteria culture to have the capacity to utilize citrate as the sole C source. Microscopic organisms that can create the compound citrase can transport the citrate into the cell and utilize it as a wellspring of C. Since the medium does not contain whatever other hotspot for C, just those microorganisms that can deliver citrase can develop in this medium. At the point when societies can utilize the citrate they separate it, producing sodium bicarbonate, which changes the pH of the medium to soluble. The pH pointer in the medium (bromothymol blue) changes to a blue shading from its unique green shading. +Slide 34
LAB 17: Carbohydrate UtilizationSlide 35
Lab 17: KIA medium C = Uninoculated 3 = Glucose fermenter + H2S maker 1 = Non-fermenter 4 = Glucose+Lactose fermenter, gas 2 = Glucose fermenter 5 = Gluc + Lact ferm + H2S makerSlide 36
Lab 17: Fermentation of Carbohydrates F-tubesSlide 37
SUGAR Fermentation Detection depends on corrosive generation because of sugar aging. The pH pointer is PHENOL RED. Phenol red turns yellow under acidic conditions. Subsequently, yellow means a positive outcome.Slide 38
The capacity to mature particular sugars is dependant on the capacity of the bacterium to deliver the particular proteins required for the transport and digestion system of that specific sugar. In this way maturation of different sugars can be utilized to describe microscopic organisms. The F-tubes utilize phenol red in the medium as pH pointer and the utilization of reversed tubes to distinguish creation of gasses. Results are recorded as Negative (no digestion system); Acid (+ response); Acid + Gas (+ with gas generation).Slide 39
LAB 18: Unknown & Single Colony Isolation Know how to utilize the Dichotomous Key to distinguish a bacterium in light of morphology, Gram recoloring, endospore generation, and different metabolic responses. See p.58-61of lab manual.Slide 41
LAB 19: Pour Plate Pour Plate Technique Serial Dilution Colony Forming Unit (CFU) Quantification of Bacteria in Cell/mlSlide 42
1 2 3 4 5 *Best 30-300 CFU = 100 Dilution = 1000 Hence 100 X 1,000 = 100,000 = 1x10 5 (1:1 1:10 1:100 1:1000 1:10,000) TMTC 1000 400 100* 20 (CFU) Lab 19: Pour Plate Dilution Series: 10 0 10 - 1 10 - 2 10 - 3 10 - 4 (Dilution) Bacteria Enumeration 1mlSlide 43
Bacteria Enumeration 1x10 - 5 1x10 - 6 Cell/ml= (CFU X weakening element)/volumeSlide 44
LAB 20: Most Probable Number MPN strategy, MPN table Durham tubes Presumptive, Confirmed, and Completed testsSlide 45
Lab 20: Most Probable Number (MPN) Bacteria Enumeration (Presumptive) MPN strategy: first - Presumptive test: development on lauryl tryptose soup second - Confirmed test: on Eosine-Methylene Blue Agar (EMB) third - Completed testsSlide 46
LAB 21: Phage Characterization and Quantification Plaque, Plaque Forming Unit (PFU) Serial Dilution, Phage evaluation T1phageSlide 47
BACTERIOPHAGE 1x10 - 4 Dilution Plaque (clear zone) 1x10 - 6 DilutionSlide 48
LAB 22: Bacterial Aggutination & ImmunoprecipitationSlide 49
Immunoprecipitation - is the response between a solvent antigen and its particular antibodies dissolvable antigens are littler and in arrangement; complexing with antibodies make these bigger and they drop out of arrangement as a hasten –visible to the eye. Counter acting agent specificity known ( poison, protein, and so on ) Antigen nearness or character not known (?) Precipitation �� reaction amongst neutralizer and dissolvable antigenSlide 50
Immunoprecipitation Antibody specificity known ( poison, protein, and so forth. ) Antigen nearness or character not known (?) Precipitation �� reaction amongst counter acting agent and dissolvable antigenSlide 51
Immunoprecipitation Reaction of personality Reaction of nonidentitySlide 53
LAB 23:Staphyloslide Latex Test Kit Example of AgglutinationSlide 54
Bacterial agglutination S. aureus S. epidermidis agglutination no agglutination ?Slide 55
Bacterial agglutination ( new technique ). S. aureus S. epidermidis #1 Mark your bacterial agglutination cardsSlide 56
Bacterial agglutination S. aureus S. epidermidis #2 Mix the latex
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