Quiz 4 Flashcards
Quiz 4 transformations and media types
MacConkey Agar (MAC)
- Selective and differential
- Useful in ID of Enterobacteriaceae (Gram-)
- Clinical laboratories
- Bile Salts, Crystal Violet: Inhibits Gram+
- Neutral Red dye: Stains microbes that ferment lactose. Ph is lowered so they get stained a pink color.
- Escherichia coli will grow red/pink colonies because it will use the lactose in the media.
- Non-fermenters will grom white/colorless because they used peptoe instead of lactose. This produces ammonia.
Eosin Methylene Blue Agar (EMB)
- Selective and Differential
- Inhibits Gram+ and Selects Gram-
- differentiates coliform and non-coliform GNRs
- Used to test quality of water. Not used in a lab setting
- Eosin Y Methylene blue: Ph indicator forms dark purple precipitate at low Ph due to fermentation
- Lactose: Coliform will ferment the lactose
- Escherichia coli has a strong fermentation and precipitation of acid and indicator complex. This will case it to produce a green metallic sheen.
Coliform
- GNR and ferment lactose
- Escherichia coli
- Enterobacter
- Serratia
- Citrobacter
CHROMOGENIC MEDIA
- ID of certain bacteria
- Chromogens: convert to different colors when degraded by different microbe’s enzymes
- Rapid detection of MRSA
- New ones created for drug resistant pathogens like Escherichia coli, Candida
- HardyCHROM UTI: used for urinary tract pathogens
FERMENTATION AND UTILIZATION MEDIA
Help identify bacteria based on:
- Sugar they utilize for fermantiation
- the acids they produce
- Ability to utilize O2
PHENOL RED BROTHS
(Durham Tube Sugar Fermantations)
DEXTROSE, LACTOSE, SUCROSE (GNR)
- Distiguishes carbohydrate fermenters from non fermenters.
- Distiguishes which carbohydrate was used by the products formed
- Media: 0.5-1% Carb broth. Dextrose, Lactose or Sucrose
- Pepton with Phenol red: The inverted Durham tube for gas detection
- Carb fermentation results in Acid. Those may also produce Gas.
- Gas production= gas bubbles in Durham tube
- Acid= Color changed form red to yellow
METHYL RED TEST (MR)
Mixed Acid Fermentation
Determines mixed acid fermentation
Part of IMVic tests
MRVP broth: Media of pepto glucose broth used for this test and the VP test
Methynol Red: Added after incubation for pH indication
VOGES PROSKAUER TEST (VP)
Butanediol Production
- Detects production of acetoin or butaneidol from fermantation of glucose
- part of IMVic test
- MRVP media: glucose pepto
- Barrit’s reagents A (naphthol) added after incubation
- Barrit’s reagents B (potassium hydroxide, KOH) added after incubation
CATALASE TEST
- Detects the production of the anzyme Catalase
- TSA media
- Reagent: 3% Hydrogen Peroxide (H2O2)
- Hydrogen Peroxide is a by-product of oxidative repiration. Catalase converts Hydrogen Peroxide to Oxygen and Water. Anaerobes lack this enzyme
OXIDASE TEST
- Detects the production of enzyme cytochrome or oxidase
- We used oxidase dry slides
- Organisms that use oxygen will have the cytochrome oxidase transfers electrons to the oxygen.
- Thre reagens in dry slide will change from yellow to purple when oxidized
CITRATE
Determine ability to use citrate as sole source of carbon.
Part of IMVic tests
Media: Simmons Citrate Agar. Contains sodium citrate as sole carbon source, mineral salts and pH Bromothymol blue.
Bromothymol blue: pH indicator
Utilization of citrate leaves a sodium residue. This increases pH
GNR
- Escherichia coli
- Serratia marcescens
- Pseudomonas fluorescens
- Enterobacter aerogenes
- Alcaligenes viscolactis
- Citrobacter freundii
LYSES
Rupture of cell wall of membrane
LYTIC
It causes lysis
LYTIC CYCLE
Two basic phage infection cycles:
- Lytic cycle
- Lysogenic cycle
SOFT AGAR OVERLAY
- 1% Agar
- Soft agar in a tub, kept in hot water bath
CONFLUENT LAWA OF GROWTH
When the growth of bacteria covers entire surface of media
PLAQUES
- Space where the bacteria have been lysed by the bacteriaphage, their cells are broken down and no longer visible.
- Clear spots
TRAMSFORMATION
When the bacteria takes plasmid DNA from the surrounding environent.
CONJUGATION
Bacteria transfer genes from one bacterium to another
GENE REGULATION
Expressing the gene that was obtained through transformation
GENETIC ENGINEERING
Forcing a bacterium to take in a plasmid DNA from the surrounding and express that gene. It would not normally have that gene.
pAMP
Plasmind that contains a gene that provided Escherichia coli with Ampicillin resistance
RECOMBINANT PLASMIDS
- Plasmids constructed form genes of different organisms
- In a lab, plasmids can be combined with other DNA plasmids.
- For example, combining antibiotic resitant gene with another gene
SELECTALE MARKERS
Used to help us differentiate the bacteria that were transformed. If we add a recombinan plasmid of a gene that allos a bacteria to be resistant to antibiotic and another gene of interest. The bacteria that groes even in the prescence of antibiotic would be the “selectable marker” because those are the bacteria that have taken both genes.
CAPSULE
- One type of glycocalyx
Structure present in some bacteria:
- Protects cell from dehydration and nutrient loss
- Inhibits killing by WBC through phagocytosis
- Aids bacteria in attachment (biofilms)
PLASMIDS
- Nonessential pieces of DNA seperated from chromosomes
- ofter confer protective traits suck as resistance to antibiotics
- Easily manipulated in a lab setting which makes them important in modern engineering techniques.
GFP
Green fluorescent protein pGLO
RECOMBINANT pGLO PLASMID
Three different gens joined together
Bla: betalactamese - makes bacteria resistant to beta-lactam antibiotics (Penicillin)
graC: Regulates expression of GFP. (expressed only in arabinose)
COMPETENT CELL
- Cells capeble of undergoing transformation by using special DNA binding receptors on their cell wall
- Cells whose cell wall can be manipulated into allowing DNA to pass through
- In the lab, we made Escherichia coli cpmpetent by using calcium chloride and heat shock
- During rapid growth it is easier to make cells competent than in other stages
SMOOTH COLONIES (S)
- Strain is virulent
- Prevents detection by immune system
ROUGH COLONIES (R)
- Strain is benign
- lacks protective capsule
- Destroyed by immune system
ARABINOSE
A sugar
In our experiment, it will regulate the expression of GFP
It will only glow in the prescence of this sugar
LOG GROWTH
Rapid cell division
LAG PHASE
Very slow growth because cell number is slow.
