Lab Exam 1 Flashcards
ubiquitous
microorganisms are always present- ie they are ubiquitous -as they are in the air, surfaces, skin clothing. Because of this, they are always a potential source of contamination during an experiment.
aseptic technique
a series of steps to prevent contamination during manipulations of cultures and sterile culture media, both liquid and solid.
Keeping things clean
culture
a liquid or solid substance that allows the growth or multiplication of microorganisms. The culture has a particular nutritive makeup and the microorganisms are grown in laboratory conditions
media
combination of nutritive substances that can be altered depending on the microorganism that is to be cultured. Different microorganisms may grow on some media but not on others due to specific metabolic or nutritive requirements
-nutrient solutions used to culture microorganisms
-may be prepared as a liquid (broth, planktonic) or solid (agar, colonies)
chemically-defined media
basal (basic) salt solution, which supplies the inorganic requirements of the organism, and organic components that provide a source of carbon and energy. The exact chemical composition of each component is known
Complex media
contain substances that are rich in both inorganic and organic nutrients such as meat or vegetable infusions, blood, and hormones. The exact chemical composition of the media is not known.
Enriched media
are loaded with nutrients and macromolecular monomers to promote the growth of even the most fastidious organisms
Minimal media
Minimal media contain only the most basic nutrients
Fastidious organisms
Are organisms that have specific nutritive requirements in order to grow.
How is culture media sterilized?
In an autoclave. This eliminates any microorganisms that may be present (as they are ubiquitous; found everywhere)
High pressure and high temperature
inoculation
the process of introducing microbes into a culture media so that it reproduces there
incubation
incubated under the desired environmental
conditions
Putting microorganism and the media its in under the desired environmental conditions to promote growth
What is a pure culture?
When there is only one microbial species present on agar or in a broth media.
Colony morphology
shape, surface, elevation, size, pigment, opacity
cellular morphology
cell shape, size, cell arrangement, Gram reaction
colony forming unit
It is assumed that each colony on the spread plate originated from a single organism from the dilution tube, so we call each colony a colony forming unit (CFU) and we measure bacterial concentration as the number of CFUs per ml of original culture
How to enumerate bacteria in a sample from CFUs?
Number of CFUs on the plate x 10 x 10^x (depends on the dilution of the plate you are counting CFUs on)
Selective media
contain compounds that selectively enrich and/or selectively repress the growth of certain organisms while not affecting the growth of others. In selective enrichment, the nutritional or environmental conditions are controlled to favor the growth of a sought-after species. Selective repression involves inhibiting the growth of interfering species while permitting the growth of a sought-after species.
differential media
contain an indicator that differentiates the
occurrence of specific chemical reactions such that groups of bacteria can be differentiated from each other based on their ability to carry out that reaction.
Acid-fast stain (Kinyoun method)
Prepare smear - flood smear with Kinyoun carbolfuchsin -10min -rinse- decolourize with acid alcohol -rinse - flood with Brilliant Green stain for 1 min
acid fast organisms are red, others are blue/green
Endospore stain
The organism is grown on sporulation agar for 48 hours before preparing an endospore stain
2. Prepare a bacterial smear
3. Place the slide on a heating rack in the fume hood. Cover the smear with a piece of paper towel
that just covers the smear and moisten the towel with Malachite Green. Steam gently for 10
minutes, keeping the paper moist with stain.
4. Remove the paper towel with forceps and rinse the slide with water to remove excess stain
5. Counterstain with Safranin for 1 minute. Rinse with water, blot dry, and observe
ENDOSPORES ARE GREEN AND THE REST OF THE CELL IS PINK/RED
protoplast
When the cell wall is removed, a viable protoplast is obtained that consists of a cell membrane and all intracellular components.
Protoplasts are produced from Gram-positive bacteria by removing the cell wall with lysozyme
spheroplast
In Gram-negative bacteria, the lysozyme will act on the peptidoglycan layer, but it does not remove the
lipopolysaccharides and lipoproteins of the outer membrane, which causes remnants of the cell wall to remain associated with the cell. These are called spheroplasts.
Endospores
Differentiated bacterial cells that form due to gradual changes in the nutritional and physical environment of the organism.
highly resistant
Can remain dormant for a long period of time
the location of the endospore in the cell is usually characteristic of the species
only formed in Gram + organisms
carcinogenic compounds
induce increased rates of mutation and as such have the potential to cause cancer
The Ames Test
Developed by Dr. Bruce Ames
The test screens for carcinogenic compounds
It really tests for mutagenicity but also for potentially carcinogenic substances
test chemicals for mutagenesis has been to measure the rate of back mutations
(reversions) in strains of auxotrophic bacteria
auxotrophic Salmonella strain for histidine
mutagenicity
The capacity to cause mutations - correlates with carcinogenesis by 85-90%
auxotrophic
A mutant organism that requires a specific nutrient to be added in order to grow.
prototrophy
Being able to obtain all required nutrients from inorganic matter. No additional organic compounds need to be added.
ie. no longer auxotrophic?
Cardinal temperatures
The range of temperatures in which an organism is able to grow/growth is observed
The temperature range of an organism is the range at which the organism can survive (not necessarily grow)
Lab rule
Eating, drinking, and smoking/vaping are prohibited in all laboratories. Cell phones
are not allowed in the laboratories.
Where does the radioactive material go?
Liquid - into a Radioactive liquid waste container
Solid - placed in the solid Radioactive waste container in the Safety office (gloves, pipettes, etc)
What goes in the blue container?
Clean plastic lab wear
Clean broken glass like broken pipettes, pasteur pipettes.
NO CHEMICAL, BIOLOGICAL, OR RADIOACTIVE MATERIALS.
What goes in the yellow container?
All sharps
Broken materials or materials that are contaminated with blood, body fluids, or biohazardous materials
All broken, contaminated glassware and plastic is placed in the yellow bucket
Where are petri plates placed??
Placed in clear biohazardous bags for autoclaving.
Where are liquid chemicals/fixatives placed?
Placed in 20L containers in the lab provided by the Safety Office
What levels of Biosafety Level designation are the materials we use in our labs
1 and 2.
The level 2 ones are capable of causing disease and pose as moderate hazards.
Level 1 - do not consistently cause disease upon exposure to immunocompromised idvls.
Go up to level 4 but we only use 1 and 2
Planktonic
Microorganisms are planktonic if they are free-living in a liquid broth media (not attached to a surface)
Crystal violet
Dyes cells purple
Crystal violet
Dyes cells purple
ETHANOL (or alcohol)
decolourizes gram negative cells
Iodine purpose in Gram staining
Iodine fixes the crystal violet into the cell wall of the bacteria by working as a mordant. A mordant forms a complex that adheres tightly to the cell wall. Gram-positive cells will remain purple because of this adherence to the iodine
Order of things in Gram Staining
Crystal Violet for one minute - rinse with water - Iodine for one minute - rinse with water. Decolourize with 95% ethanol for 10 seconds - alternate with water. Flood with Safranin for one minute. Rinse with water and blot dry carefully.
Safranin
Counter stain used during the last step of Gram staining. It dyes the decolourized Gram negative cells pink. The gram positive cells do not appear pink because they held the darker purple stain from the crystal violet.
MacConkey
Crystal violet and bile salts inhibit growth of Gram Positive Organisms
Those that ferment lactose will cause the colour to change due to a pH indicator in the agar.
SF agar
differentiation of enterococci from streptococci
Enterococci ferment dextrose and grow in the presence of the inhibitor
sodium azide (yellow-brown colour if ferment dextrose)
No colour change if do not ferment dextrose
No gram negative bacteria grow
Mannitol Salts Agar
Contains sodium chloride at 7.5%. only Staphylococci will grow in these conditions
Coagulase-positive staphylococci
produce growth of yellow colonies with yellow zones. Coagulase negative staphylococci produce small red colonies with no color change to the medium. Micrococcus produce large, white to orange colonies, with no color change to the medium. Most other bacteria will be inhibited (incl Gram neg)
Colour change occurs due to the fermentation of mannitol (aids in the differentiation of Staphyloccci)
Acid-fast Stain (Kinyoun method)
Prepare a bacterial smear
Flood smear with Kinyoun carbolfuchsin for 0 mins
rinse with water
Decolourize with acid-alcohol
rinse with water
Counterstain with brilliant green for 1 min
rinse with water, blot dry
ACID FAST ORGANISMS ARE RED; OTHERS ARE BLUE/GREEN
Are acid fast organisms red or blue/green?
ACID FAST organism are red while others are blue/green
Endospore staining
Bacteria grown on sporulation agar for 48 hours first
Prepare a bacterial smear and place on heating rack in fumehood
Place paper towel and keep moist with Malachite Green. Steam gently for 10
minutes, keeping the paper moist with stain.
rinse after. Counterstain with Safranin for 1 minute. Rinse with water, blot dry, and observe
ENDOSPORES ARE GREEN AND THE REST OF THE CELL IS PINK/RED
What colour are stained endospores?
ENDOSPORES ARE GREEN AND THE REST OF THE CELL IS PINK/RED
Endospores only form in Gram positive organisms
reversions
back mutations- mutating back to a prototrophic phenotype
Standard way to test for mutagenisis
zone of inhibition
The diameter of the ring where the organisms are not capable of growing,
including the disc itself
cidal
An antimicrobial substance which is concerned with the killing of microorganisms
static
An antimicrobial substance which inhibits growth of an organism
lytic
An antimicrobial substance which lyses the microorganism cell
antiseptic
cidal, static, or lytic against microorganisms but are still safe enough for use on living
tissues.
disinfectants
are more potent cidal or lytic agents that destroy nearly all microorganisms but
can be applied only to inanimate material because of their toxicity
cidal
phenol coefficient
the ratio of the test agent’s effectiveness to the effectiveness of phenol
If the coefficient is greater than 1, the test disinfectant is better than phenol; if the coefficient is less than 1, the test disinfectant is not as effective as phenol
(reciprocal effective dilution of test disinfectant)/(reciprocal effective dilution of phenol)
phenol
the first disinfectant so the phenol coefficient describes how potent a disinfectant is relative to phenol
effective dilution
the dilution of agent that completely inhibits growth at 10 minutes exposure, but not at 5 minutes exposure
MAC sugar
lactose
MS sugar
mannitol
SF agar sugar
dextrose
the substance used as a positive control in the Ames test
sodium azide
microscope slides
Go into the used slide container
the substance used as a negative control in the Ames test
sterile water
lymphadenopathy
A symptom that distinguishes monkey pox from other viral zoonotic diseases
