Midterm Flashcards
-DNA extraction: Step 1
1st step: break the cells open to expose the DNA, can use mortar pestle
-DNA extraction: Step 2
2nd step: remove lipids and proteins with detergent, distilled water, pure water, glycerol
-DNA extraction: Step 3
3rd step: precipitate the DNA with alcohol
aseptic techniques:
No eating or drinking in the lab
Wiping surfaces with disinfectant/alcohol
Not growing microorganisms at body temperature
Using sterile loops when transferring cultures
Flaming culture bottle necks to prevent contamination
Sterilizing (using an autoclave) or disposing of all used equipment
Cleaning and disinfecting lab surfaces prior to use
Limiting the duration that cultures or media are uncapped and exposed to air
Keeping petri dishes closed whenever possible
Effectively sterilizing inoculating loops and other equipment that comes into contact with cultures or media
Avoiding breathing on cultures or sterile instruments
Microscope
devices that allow the observer to an exceedingly close view of minute particles
Laminar flow hood
closed device primarily for processes or instruments sensitive to microbial contamination
Autoclave
is a pressurized chamber used for the process of sterilization and disinfection by combining three factors: time, pressure and steam
petri dish
a thin layer of agar is poured in the bottom of the plate and is stored upside down so condensation does not land in the agar
Slant
– agar is poured in a test tube and then allowed to solidify in the test tube at an angle
Analytical Balance
type of balance that is commonly used for the measurement of mass in the sub-milligram range.
Incubator
device that is used in the laboratories for the growth and maintenance of microorganisms and cultures
Hot Plate
stand-alone appliance used in microbiology laboratories as a tabletop heating system.
Magnetic Stirrer
is a device commonly used in microbiology laboratories for the purpose of mixing liquids.
Water Bath
conventional device that is used for chemical reactions that required a controlled environment at a constant temperature.
Bunsen Burner
a standard tool used in laboratories, named after Robert Bunsen.
It is a gas-fueled single open flame.
Spectrophotometer
is an optical instrument for measuring the intensity of light in relation to the wavelength.
Based on the amount of light absorbed by a colored solution, a quantitative analysis of the solution can be done.
Centrifuge
device that allows the rotation of an object about a single axis, where an outward force is applied perpendicularly to the axis.
Microcentrifuge
Same as centrifuge but for smaller quantities
Thermocycler or PCR Machine
This machine cycles through multiple temperatures to complete the polymerase chain reaction
The typical cycle goes from 96ºC to 58ºC to 72ºC
These temperature cycles are then repeated up to 40 times
Blender
Typical household appliance that is used to blend ingredients for making media
Vortex mixer
is one of the basic technologies used for the mixing of samples in glass tubes or flasks in laboratories.
Pipettes
Use to transfer material in specific amounts
Serological Pipet – plastic or glass cylinder marked with specific volumes, can move less than a ml to over 100 ml
Pasteur Pipet – plastic or glass tube with an attached bulb without specific volumes marked
Micropipet – tool that specific delivers an amount between 0.1 μl to 1000 μl
Glassware
Test tube Microcentrifuge tube (plastic) Erlenmeyer flask Beaker Media bottle Graduated Cylinder Spreader
Other LAB equipment
Petri Dish
Inoculating Loop
Optical Microscope, Pathway of Light
Light source, Diaphragm, Condenser, Objective Lense, Ocular Lense, Eye
Total Magnification =
Power of Objective x Usual Power
Resolution or RP=
wavelength of light measured in nm/2x numerical aperture of objective lense
Bright-field
le. Bright-field microscopy is the simplest of a range of techniques used for illumination of samples in light microscopes, and its simplicity makes it a popular technique. The typical appearance of a bright-field microscopy image is a dark sample on a bright background
Dark-field
Dark-field microscopy describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen is generally dark.
Fluorescent Microscopy
an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances.
Phase Contrast Microscopy
optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations.
Confocal Microscopy
an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation
Scanning Electron Microscope
is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons
Group I Virus, Mode of mRNA production, Example
Double Stranded DNA, mRNA is transcribed from DNA template, Herpex simplex
Group II Virus, Mode of mRNA production, Example
Single Stranded, DNA is converted to double stranded form before RNA is transcribed, Canine parvovirus
Group III Virus, Mode of mRNA production, Example
Double Stranded RNA, mRNA is transcribed from the RNA genome, Childhood gastroenterovirus, rotavirus
Group IV Virus, Mode of mRNA production, Example
Single stranded RNA (+), Genome function as mRNA, common cold, picornavirus, coronovirus
Group V Virus, Mode of mRNA production, Example
Single stranded RNA (-), mRNA is transcribed from the RNA genome, Rabies, rhabdovirus
Group VI Virus, Mode of mRNA production, Example
SIngle stranded RNA viruses with reverse transcriptase, Reverse transcriptase makes DNA from the RNA genome; DNA is then incorporated in the host genome, mRNA is transcribed from the incorporated DNA, HIV Human immunodeficiency virus
Group VII Virus, Mode of mRNA production, Example
Double Stranded DNA Viruses with reverse transcriptase, The viral genome is double stranded DNA, but viral DNA is replicated through and RNA intermediate; the RNA may serve directly as mRNA or as a template to make RNA, Hepatitis B Virus hepadnavirus
Enterobacter
genus of common Gram-negative, facultatively anaerobic, rod-shaped, non-spore-forming bacteria of the family Enterobacteriaceae.
Escherichia
a genus of Gram-negative, non-spore-forming, facultatively anaerobic, rod-shaped bacteria from the family Enterobacteriaceae.
Gram Staining
Differentiates Gram-negative and Gram-positive bacteria due to the difference in the cell wall morphology
Alveolates – Eukaryotic
Apicomplexans (sporozoan) (parasite) (Protist)
Dinoflagellate
Ciliates
Fungi – Eukaryotic
Ascomycota (sack like structure)
Basidiomycota (mushrooms, puffballs)
Zygomycota ( bread molds)
Stromatolites (Diatoms – Eukaryotic, plankton)
Bacillariophyceae (Algae)
Archaeplastida
Chlorophyta ( green algae)
Rhodophyta (red algae)
Charophyta (algae)
Phaeophyta (brown algae)
Ameoba (eukaryotic) Protist
Cercozoan (single celled)
Excavates- Eukaryotes unicellular
Diplomonads (parasitic flagellates) Parabasilids (flagellated protist subgroup) Euglenazoa (parasitic flagellates) Euglenid (same as euglenazoa) Kinetoplastida (flagellated protist)
Rhizaria
Foraminiferans (single celled, with shells, protist)
Radiolarians (shelled animal)
Flat-Worms (animals)
Nematoda (parasitic)
Platyhelminthes (Ocean colorful worm)
Salmonella
is found in MacConkey’s Agar. It is generally used for differentiating strains of Salmonella typhosa from members of coliform group; however, the medium supports the growth of all salmonella and SHIGELLA strains and gives good differentiation between these enteric pathogens and the coliform groups. Brick red
Shigella
is found in MacConkey’s Agar. It is generally used for differentiating strains of Salmonella typhosa from members of coliform group; however, the medium supports the growth of all salmonella and SHIGELLA strains and gives good differentiation between these enteric pathogens and the coliform groups. Brick red
Staphylococcus
is found in (MSA) Mannitol Salt agar, The high salt concentration inhibits the growth of most bacteria other than staphylococci. On MSA, pathogenic Staphylococcus aureus produces small colonies surrounded by yellow zones.
Nutrient Agar or Broth (NA & NB)
This media is a general-purpose media used for non-fastidious microbes including many BACTERIA and FUNGI.
Potato Dextrose Agar (PDA)
This media is a general-purpose media used for FUNGI
Trypticase Soy Agar (TSA)
This media is a GENERAL-PURPOSE media used for BACTERIA.
Mannitol Salt Agar (MSA)
This Selective and differential media is sued to isolate STAPHYLOCOCCUS. Yellow zones, due to acid changes the indicator color from red to yellow
Methylene Blue Agar (EMB)
This selective and differential media is used to isolate ENETERIC BACTERIA THAT HYDROLYZE UREA. Also does not grow Gram Positive, only GRAM NEGATIVE. E.coli green color, Enterobacteria, PINKISH color. Ferments sugar and produces colored colonies, lactose
MacConkey’s Agar
This selective and differential media is used to isolate enteric bacteria that FERMENT LACTOSE. Won’t grow gram positive, only GRAM NEGATIVE. Bile and brick red.
Salt Agar
Algae, semi-solid, high salt content
Hay Infusion
Paramecium and other Protozoans, Alfalfa Media
Live Cell Culture
Virus, live host cells, obligate parasites, use host cells on the media.
Gram positive
- color is purple/blue, thick peptidoglycan cell wall.
Gram negative
- color is red/pinkish, thin peptidoglycan cell wall.
• Polymerase Chain Reaction PCR
Phases of PCR:
1. Denaturation Phase: raises temperature to break the DNA into single strands. Best at 92 or 95 Celsius
2. Annealing Phase: allows for primers to form hydrogen bonds with the single stranded DNA best at 58-60C Hydrogen bond
Brownian motion hydrogen bond
3. Extension Phase: adds complementary nucleotides to single strand of DNA
58-62C Highly specific
54-57C
>54C not specific
-PCR reagent indicates what part should be replicated: primers
-PCR reagent that provides nucleotides/base pairs used o make copies of DNA: dNTPs
Taq polymerase works best at 72C
Real time PCR tells you what organism you have or working with Bacteria or Viruses
BLAST Basic local alignment search tool
when identifying a bacteria using BLAST which metric is used (Percent Identity/similarity)
Most widely used bioinformatics tool, a computer algorithm to help identify the entity of our sequences.
Query Coverage- how much your sequence was used to MATCH to the EXISTING DATABASE
Percent similarity- Bacteria 99% or high to match
• General media for all microbes
(TSA) Trypticase Soy Agar- can grow mostly anything
• -EMB: (Eosin Methylene Blue Agar)
Enterobacter aerogenes=pink
Colonies of E. coli= green
Selective for gram-negative intestinal bacteria
Lactose
-MSA: (Mannitol Salt Agar)
Selective for staphylococcus
Bacterium ferments in mannitol=yellow
Goes from red to yellow
MacConkey’s:
Selective for salmonella
Coliform bacteria make colonies=red
Crystals inhibit gram-positive pathogens
• Staining-Positive
can be either acidic or basic (vast majority basic) dye will go into the cell envelope and stain the border while acidic will be more dispersed (cell will be colored by the dye)
• Staining-Negative
all acidic dyes, dyes stay outside of the cell and dye backgrounds outside of the cell (clear or transparent in appearance)
Staining: Simple
just use one dye (ex. methylene blue, basic fuchsin, crystal violet)
Staining: Differential
use two different color stains to differentiate between two different types of cell morphologies/identities. Nigrosine or India ink/ acidic dyes
Gram Staining
Differentiates Gram Negative and Gram Positive Bacteria due to the difference in cell wall morphology
Crystal violet-primary stain
Iodine-mordant
Decolorization with Alcohol
Safranin-secondary stain
Endospore staining
indicate that bacterial cell can survive under environmental stress (DNA within and form an extra layer of cell membrane around chromosome) identify whether we have endospores or vegetative cells (regular bacterial cell)
Schaeffer-Fulton Method- Malachite green is the primary stain (endospore cell) green color
Safranin is secondary stain (vegetative cell) red color
Acid-Fast Staining
used to identify the genus Mycobacterium
Mycobacterium cause diseases such as tuberculosis and leprosy
Mycobacterium have a waxy material in their cell wall lipids called mycolic acid
Acid Fast Staining- Kinyoun Method
Fuchsin- primary stain (acid fats cells in red)
Heat- mordant
-Decolorized by acid alcohol
-Methylene blue- secondary stain (non-acid fast cells in blue)
