BIO 220 LAB Practical Exam 1 CSUSB Lujan
Eosin Methylene Blue Agar (EMB)
**Selective/Differential** Selective for: Gram (-) **Components:** Eosin dye/MB dye; Lactose Results: **Differentiates** between lactose and non lactose fermentors: - ( Some acid = pink ) - ( Large acid = metallic green ) - ( No acid = colorless ) (E. aerogenes = Pink, E. coli = Green (Lactose fermentation), Pseudomonas sp = Clear.)
Gram stain
- ex.3 used crystal violet(primary stain) and iodine(mordant), washing w/ decolorizer, counterstaining w/ safranin. -Results: gram+ bacteria= purple dye - gram- organisms having it decolorized so that the pink counterstain shows up. -used K. pneumo (bacilli, G-, encapsulated) & S. aureus (cocci, G+)
Aquatic environments have pathogenic members including : Bacillus & Clostridium :
-Bacillus anthracis -Clostridium: tetani, botulinum, perfringens
Capsule: ○ Made of :
-Protective layer surrounding some bacteria cells; usually means its more likely to be virulent (eg. evade WBC) -Made of polysaccharides & polypeptides → stain resistant -Looks like a white halo when the cells get stained
Negative stain
-colors the background, which makes capsules more visible -determine basic morphology and arrangement of cells -NOT HEAT FIX - (ex.2 india ink) - used K. pneumoniae
Gelatinase Test
-detects enzymatic breakdown of gelatin to polypeptides -Organisms which produce the enzyme gelatinase cause the media to LIQUIFY + ; solid-
Capsule stain
-determine bacteria surrounded by protective capsule -congo red (acidic), spreader slide, Manual's (basic), AIR DRY
*SPREAD* Plate Method of Isolation
-diluted microbial sample is deposited on an agar plate and spread uniformly across the surface with a plate spreader -with a properly diluted sample, cells will be deposited far enough apart on the agar surface to grow into individual colonies
acid fast stain
-identify bacteria that possess mycolic acid -smear both bacteria on slide, steam heat carbolfuchsin(primary stain)=pink -decolorize w/ acid alcohol, methylene blue(counterstain) -acid-fast=pink / non-acid=blue -Resistant to decolorization by acid-alcohols = pink!
Sterilize transfer tools
-insert loop/needle into flame before and after using it -flame the mouth of the culture tube before and after using it
*STREAK* Plate Method of isolation
A bacterial sample is streaked over the surface of an agar plate.
Endospore Stain
A differential stain used to detect the presence and location of spores in bacterial cells. -Endospore: the dormant form of the bacterium ○ Has a keratin coating → survival under harsh conditions (Chemical exposure, UV light, High heat/freezing) ○ STEAM HEAT malachite green stain ○ Vegetative cell (active form) will be decolorized and counterstained with safranin
Gram Stain
A staining method that distinguishes between two different kinds of bacterial cell walls.
Practicing basic transfers using the Aseptic Technique
Inoculations from a broth tube (& slant tube) to: broth tube, agar plate, slant tube - Used K. pneumoniae
Microscope funcitons
Ocular Lenses: put eyeballs (10X) Objective lenses: closer to specimen Revolving nosepiece: choice of lens Iris Diaphragm: below the stage Coarse Adjustment knob: large knob Fine Adjustment knob: small knob
Ways to inoculate agar plate
Straight-line streak & Zigzag streak
Colony Isolation techniques (2)
Streak & Spread Plate technique
Methyl Red Voges Proskauer (MRVP)
Tests for: MR - Fermentation of sugar with acid end-products VR - conversion of acid end-products to neutral ones
Urease Test detects:
The above test is used to differentiate organisms on their ability to hydrolyze urea using the enzyme urease.
Urease Test
Urease positive = PINK NEGATIVE (no color change)
Triple Sugar Iron Agar (TSI)
Yellow butt (acid) - ferments glucose Red Butt (alkaline) - no glucose fermentation yellow slant (acid) - lactose, sucrose or both Red slant (alkaline) - non lactose or sucrose utilization Gas: breaks in agar Black: H2S
Bacillus cereus
a bacillus shaped bacteria
Staphylococcus aureus
a cocci shaped bacteria
Simple stain
a method of staining microorganisms with a single basic dye -determine basic morphology and arrangement of cells -colors the cell (ex.1 used methylene blue) - used K. pneumoniae
Yeast
a single-celled fungus
human blood
an animal cell
Who to Obtain pure culture for Isolation technique
attaining isolated colonies which are accumulations of cells resulting from the division of a single cell
structural stains
capsule & endospore stain -used to identify and study the structure of bacteria
Phenol Red Broth
differential -yellow + bubble = fermentation with gas -yellow, no bubble = fermentation w/out gas -red, no bubble = no fermentation -pink, no bubble = degradation of peptone, alkaline
what can the products of gelatin hydrolysis be used for:
energy metabolism, new proteins(amino acids), materials for replication (amino acids & nucleic acids )
Differential stains
gram stain and acid fast stain -differentiate based on chemical composition of cell wall
Staining techniques
improve visibility and allow us to determine certain characteristics of the bacteria
When performing your transfers, remember......
just because you can't see it, that doesn't mean theres nothing there !
Identification of Eukaryotic
larger, complex, membrane-bound organelles, multiple chromosomes, mitotic division
basic dyes/ simple stains
methylene blue , crystal violet, safranin (have + charge)
Objective lenses (total) Magnifications
ocular x objective=total 4X, 10X, 40X, 100X
Morphological stains
simple stain and negative stain -provide w/ cell size, shape, and arrangement
Identification of Prokaryotic
smaller, simpler, no membrane-bound organelles, single chromosome, binary fission
Treponema pallidum
spirochete bacteria
Aseptic Technique
transfer to prevent contamination to either the bacterial culture or the media it is being transferred to.
Oxidation-Fermentation Test
used to differentiate bacteria based on their ability to oxidize or ferment specific sugars; organisms that are able to ferment and oxidize the carbohydrate will turn the sealed and unsealed media yellow throughout; organisms that are able to oxidize only will turn the unsealed medium yellow and leave the sealed medium green or blue; slow or weak fermenters will turn both tubes slightly yellow at the top; organisms that are not able to to metabolize the sugar will either produce no color change or turn the medium blue due to alkaline products from amino acid degradation
mycolic acid
waxy lipid, makes cell permeable to chemical damage, dehydration, makes certain antibiotics ineffective, allows to evade immune system
Smear prep
where you smear bacteria onto a slide and fix the bacteria so they will not wash off during staining procedures
