Micro Practical Exam (exp 1-7)
Coccus Bacillus Spiral
3 morphology of bacteria
purple colorless
After decolorizer (acetone alcohol) is added what is the color of Gram positive cells? Gram negative cells?
purple pink
After safranin is added, What color is Gram positive cells? Gram negative cells?
Zone of inhibition
Area around the antibiotic disc in which bacterial colonies do not grow
cold
Bacteriostatic effect; capable of inhibiting the growth or reproduction of bacteria but its not. capable of killing them
Disinfectant
Death to disease producing microbes Higher concentration Inanimate objects
Antiseptic
Destroy or inhibit bacterial growth Does not necessarily kill microorganisms Lower concentration On skin
100 degrees water bath 30 minutes
Duration and temperature for boiling
3-5 minutes not longer than 15 minutes
During the inoculation of the test plate how long should it be allowed to stand on a flat surface?
Acid is formed when bacteria ferments lactose producing acid and gas.
Explain what is in lactose broth that indicates the presence of acid
solid
How do you classify this medium according to physical state?
Total magnification= Magnification of eyepiece x Magnification of Objective
How do you compute for total magnification? write the formula
Asexual, budding
How does Candida Albicans reproduce?
Binary fission
How does bacteria multiply through?
15mm
How far apart should the antibiotic discs be from the edge?
Get the radius (of the widest zone of inhibition) and multiply by 2
How is the zone of inhibition measures when the diameter cannot be accurately measured?
1 minute
How long should acid alcohol be on for acid fast staining?
5 minutes
How long should carbon fuschin be on for acid fast staining?
1 minute
How long should crystal violet be on the glass for gram staining?
45 seconds
How long should methylene blue be on for acid fast staining?
few seconds
How long should the acetone alcohol be on for gram staining?
16-18 hours may exceed to 20-24 hours
How long should the agar plates be incubated after inoculation in Anti-Microbial susceptibility testing?
1 minute
How long should the gram's iodine be on for gram staining?
15 seconds
How long should the safranin be on for gram staining?
upside down at 37 degrees celcius for 24 hours
How should the plate be incubated?
Nutrient broth
Identify the medium used in the tube labeled control
Candida Albicans
Identify the organism pointed
Coccobacilli
Identify the specific morphology
Diplococci
Identify the specific morphology
Spirillum
Identify the specific morphology
Streptobacilli
Identify the specific morphology
Streptococci
Identify the specific morphology
Vibrio
Identify the specific morphology
Staphylococcus
Identify the specific morphology where the pointer lies
Endospore
Identify the structure pointed by the red arrow
100 degrees celcius
Identify the temperature and time the tube labeled B is subjected to?
presence of bubbles and displacement of the medium in the small, inverted tube
In bacterial growth, what indicates gas production?
Gram negative because it shows a pink/ red color after counter staining
Is E.coli gram postive or gram negative? Why?
Autoclave
Most common method for sterilization
Germ tube
Outpouching of cw becoming tubular no constriction of base
Nosepiece (revolving)
Part of the microscope housing the objective
to obtain a reproducible result
Purpose of 15-15-15 rule
- grow and isolate all bacteria present in an infection - determine which of the bacteria that grew are most likely causing the infection and which are likely contaminants are colonizers. - obtain sufficient growth of clinically relevant bacteria to allow identification and characterization
Purpose of cultivation
Aldehydes Ethylene oxide
Sterilizing agents
producing chains or links of sausages: pseudohyphae
Structure or morphology of candida when few buds fail to detach
TRUE
TRUE OR FALSE Spirillum are all gram negative
TRUE
TRUE OR FALSE The surface appearance of the colonies can be described as opaque.
FALSE
TRUE OR FALSE This medium can grow fastidious organisms
TRUE Except Mycobacteria Bacillus Atinomyces and Streptomyces Nocardia Clostridia Corynebacterium Listria Lactobacillus Erysipelothrix
TRUE OR FALSE All bacilli are gram negative
TRUE
TRUE OR FALSE Are spirochetes unstainable by gram stain?
TRUE Except Neisseria spp Branhamella spp Moraxella spp Veillonella spp
TRUE or FALSE COCCI are ALL Gram Positive
Molecular Weight
The germicidal action of this disinfectant is correlated with its __________
1. susceptible 2. intermediate 3. resistant
The zone of inhibition is translated into? (3)
non-susceptible (NS)
Translation when the antimicrobial agent is not under resistant of intermediate
sensitive/susceptible
Translation when the antimicrobial agent may be an appropriate choice for treating the infection
Resistant
Translation when the antimicrobial agent may not be an appropriate choice for treating infection
Intermediate
Translation when the antimicrobial agent may still be effective but usually at higher concentrations
Susceptible Dose Dependent (SDD)
Translation when the antimicrobial agent would depend on the clients regimen of antibiotics
Ziehl- Neelsen Method
What acid fast staining method is used to stain this?
Lactic Acid Lactose
What acid is produced during lactose fermentation?
Mueller-Hinton agar
What agar plates were used for the antimicrobial susceptibility test?
The source culture is dead, they generally look the same as live bacteria, so you cannot really assume that cells on an agar surface or broth are alive.
What are some reasons that the experiment could possibly be unsuccessful?
1. Yeast 2. Pseudohyphae 2. Hyphae
What are the 3 morphologies of Candida Albicans?
Resistance of microbe Contamination Special characteristic of microorganism (ex, flagella)
What are the causes of unclear zones?
Food of bacteria Indicators Inhibitory substances for certain bacteria (dyes antibiotics) Control pH
What are the components of culture media?
Heat fixation Crystal Violet Dye - Primary stain Iodine - Mordant Acetone alcohol - Decolorizing solution Safranin - counterstain
What are the steps in Gram staining?
Hydrogen Carbon Dioxide
What are the two gases that are present in the tube labeled CTRL?
2% sodium carbonate
What can be added to B to increase the killing effect?
pink
What color presence of acid?
Antimicrobial
What destroys microorganisms?
Antibacterial
What destroys or inhibits bacterial growth
CO2 Hydrogen
What gas is produced during lactose fermentation?
slower diffusion false resistance
What happens when the agar is too dry in the Anti-Microbial susceptibility testing?
smaller zone sizes (false resistant) lower antimicrobial concentration
What happens when the agar is too thick in the Anti-Microbial susceptibility testing?
larger zone sizes (false sensitive/susceptible) higher antimicrobial concentration
What happens when the agar is too thin in the Anti-Microbial susceptibility testing?
Faster diffusion False susceptibility
What happens when the agar is too wet in the Anti-Microbial susceptibility testing?
Won't get the desired outcome or the culture won't grow.
What happens when the nutrient broth is only refrigerated for an hour or two and not overnight?
There is possibility that there is too much primary stain - crystal violet causes inaccuracy because it becomes gram positive even though it's supposed to be gram negative
What if after you decolorize then counterstain, it still remains purple, based on the procedure, what could be the error? What step?
Turbidity
What indication of growth is evident in A
impregnation is to cause a material to be filled or soaked with something A filled or soaked filter paper disc. inoculum
What is an Impregnated filter paper disc? impregnation is to cause a material to be filled or soaked with something. A filled or soaked filter paper disc. inoculum
Close streaking: Multiple overlapping technique OR very close streaking while turning the plate 60 degrees celsius in three different directions
What is another way of stating the type of streaking done aside from lawn streaking?
Air drying- to preserve bacterial morphology
What is done before this procedure and what is its purpose?
rapid cooling
What is done to prevent damage to other necessary proteins in classic pasteurization?
1% Barium Chloride 1% sulfuric acid reaction of both chemicals produces a fine precipitate enabling comparison visually
What is in Barium sulfate?
andrades indicator
What is in lactose broth that appears pink in the presence of acid?
1. Use of inoculum within 15 minutes of preparation 2. Application of the discs within 15 minutes of inoculating the plates 3. Starting incubation within 15 minutes of the application of disks
What is the 15-15-15 rule?
Bacillus/ Bacilli
What is the basic morphology of this organism?
Kirby-Bauer Chart
What is the chart used to determine the translation of zone diameter of the zone of inhibition?
blue
What is the color of B. subtilis?
red
What is the color of e.coli?
red
What is the color of m. tuberculosis?
purple
What is the color of s.epidermidis?
acid alcohol
What is the decolorizer used for acid fast staining?
Acetone Alcohol
What is the decolorizer used for of gram staining?
rod shaped may be: snapping- v shaped slipping- side by side
What is the description of Diplobacilli?
round to ovoid coccus in pairs
What is the description of Diplococci?
S shaped bird in flight arranged as a single flagellum at one or both poles
What is the description of Spirrilum?
spherical Grape like clusters
What is the description of Staphylococci?
rod shaped in chains arranged in small clumps
What is the description of Streptobacilli?
round to ovoid coccus in chains
What is the description of Streptococci?
comma shaped rods arranged with flagellum at one cell pole as well as pilli
What is the description of Vibrio?
short, thick, oval shaped arranged in singles or pairs
What is the description of coccobacilli?
Complete inhibition of growth
What is the end point of all reading systems? Disregarding tiny colonies which can be detected only by very close scrutiny.
Methylene blue
What is the flood counter stain used on acid fast staining?
Safranin
What is the flood counterstain of gram staining?
Gram's Iodine
What is the flood mordant of gram staining?
Crystal Violet
What is the flood primary stain of gram staining?
Colony
What is the indication of growth on this medium?
um
What is the measurement of bacteria?
Protein coagulation
What is the mechanism of action of this disinfectant?
Hucker's Method
What is the method used in gram staining?
gram-negative rod shaped bacteria
What is the morphology and shape of E. Coli?
Yeast Cell
What is the morphology described as oval-shaped and may sometimes be budding?
Acid fast positive rod- shaped bacteria non motile
What is the morphology of M. tuberculosis?
Gram-positive rod shaped
What is the morphology of S. subtilis?
Gram positive cocci grape like clusters non hemolytic on blood agar non-motile
What is the morphology of s. epidermidis?
Carbon Fuschin
What is the primary flood stain for acid fast staining?
determine whether the bacterial isolate is capable of expressing resistance to the antimicrobial agents selected for treatment
What is the primary goal of Antimicrobial Susceptibility Testing?
1. not make image blurry 2. help bridge the gap between refractive index and glass slide 3. immersing oil with objective= prevents dispersion of light rays making it focus more
What is the purpose of adding oil when using OIO
To obtain an even and complete distribution or inoculum
What is the purpose of doing close streaking?
Used on equipment that cannot be wet and on material that will not melt, catch fire, or change form when exposed to high temperatures.
What is the purpose of hot air sterilizer?
to prevent wash off during staining and allow specimen to adhere
What is the purpose of this procedure?
Staphylococcus Aureus
What is the representative organism of A?
Bacillus Subtilis
What is the representative organism?
Campylobacter jejuni
What is the representative organism?
Escherichia coli
What is the representative organism?
Mycobacterium Tuberculosis
What is the representative organism?
S. Pyogenes
What is the representative organism?
Staphylococcus aureus
What is the representative organism?
Streptococcus pneumoniae
What is the representative organism?
V. Cholerae
What is the representative organism?
15 mm for edge 20mm apart discs
What is the special arrangement of the antimicrobial discs on the agar?
Very close streaking
What is the streaking done by turning the plate each time at 60 degrees celcius
10x100= 1000
What is the total magnification
10x40= 400
What is the total magnification of this?
Inverted Durham Fermentation tube
What is used to detect gas production in CTRL
McFarland Test
What is used to standardize the approximate number of bacteria in a liquid suspension by comparing the turbidity of the test suspension?
Gram-positive species with several adaptive mechanisms for competition and environmental stress
What makes Bacillus subtitles the best bacterial suspension for this experiment? (Growth inhibition and destruction of bacteria by physical agents)
The cell wall of E. coli, a Gram-negative bacterium, play a critical role in modulating antibiotic resistance.
What makes Escherichia coli the best bacterial suspension for this experiment? (Growth inhibition and destruction of bacteria by physical agents)
E. Coli
What organism was used in experiment 6 (Action of disinfectants on microorganisms) ?
Antimicrobial Susceptibility Testing
What procedure is done to produce antimicrobial susceptability profiles and detect resistant to therapeutic agents?
Contrast staining
What quality that you need to make object stand out from Background?
4mm
What should be the depth of the agar in AntiMicrobial susceptibility testing?
Gram Stain
What stain is used for this specimen? (LPO)
Gram staining
What staining was done for this experiment?
simple streaking
What streaking is done to inoculate the swab on the agar?
Durham Tube
What tube is used to detect production of gas by microbes?
Tap water, in this experiment. No. But if you were boiling a utensil distilled water is preferred because hard water might result in the formation of a film of calcium salts on the instruments.
What water is used for the water bath in Boiling? Does type of water affect the outcome?
Ampicillin Gentamicin Penicillin Cholamphenicol Trimothoprim- Sulfamethoxazole
What were the 5 antibiotics used for the antimicrobial susceptibility test?
Enterobacteriaceae Staphylococcus Aureus Pseudomonas Aeruginosa
What were the pathogens tested for the antimicrobial susceptibility test?
Result to many dead cells contaminating the preparation Cells consume available nutrients, excrete toxic metabolites, entire population dies
What would happen if incubation exceeded 16-18 hours in Anti-Microbial susceptibility testing?
Red Colorless
When acid alcohol is used what color is Acid fast positive bacteria? Acid fast negative bacteria?
Red Blue
When using methylene blue, what colors are? Acid fast positive bacteria? Acid fast negative bacteria?
Biosafety Cabinet Level II
Where should smears for this be done?
Thin cell walls
Why are spirochetes difficult to gram stain?
To further distinguish gram + and gram - bacteria Gram + -> Purple Gram - -> Pink
Why do we have to decolorize then counterstain again?
Cell walls contain long chain mycolic acids rendering cells resistant to decolorization
Why does acid fast positive cells not decolorize when used with acid alcohol?
Sensitive bacteria are killed, but resistant germs may be resistant and grow and multiply Mutation of organism
Why does bacteria develop resistance to antimicrobial agents?
The cell wall is composed of a thin peptidoglycan layer and an outer membrane.
Why is E. coli stained negative?
peritrichous flagella
Why is e.coli motile?
Can kill microorganisms because of its ability to penetrate microbial cells. denaturing their proteins (causes proteins and enzymes to lose their three-dimensional functional shape). also may melt lipids in cytoplasmic membranes.
Why is moist heat more effective than dry heat?
gives satisfactory growth of the majority rapidly growing pathogens Not antagonistic (does not harm) the major antimicrobial agents Isotonic with blood (can be supplemented with 5% defibrinated blood for testing fastidious organisms
Why is the Mueller Hinton Infusion Agar used for the medium of choice in the AntiMicrobial suscptibility testing?
to keep bacterial population growing
Why should bacterial cultures be periodically transferred or sub cultured or to a new media?
to avoid having the discs develop resistance to the antibiotics due o pr
Why should incubation happen for 16-18 hours for the Anti-Microbial susceptibility testing?
To avoid overgrowth of bacteria
Why should it not exceed 15 minutes after inoculation in Anti-Microbial susceptibility testing?
to prevent zones overlapping
Why should the antimicrobial discs be spaced out 20 mm?
To avoid having the disc diffuse or spread right away To obtain an even and complete distribution of inoculum over the entire plate (petri dish)
Why should the plate be flat for 3-5 minutes during the Anti-Microbial susceptibility testing?
it may allow to enter the cell wall
Why shouldnt exposure to carbon fuschin be prolonged?
Used to detect production of gas by microorganisms. Small tubes places upside down in a test tube that can collect any carbon dioxide produced as a waste product of fermentation
Why was Durham's tube used in this experiment? (Action of disinfectants on microorganisms) What's the rationale?
gives satisfactory growth of the majority of rapidly growing pathogens not antagonistic to the major microbial agents
Why was Mcfarland Barium Sulfate no 0.5 used?
Crystal Violet Iodine Complex
complex formed for gram positive cells enhancing the bond between the cell wall and primary stain
120 degrees for 15 minutes under 15 lbs of pressure
duration and temperature for autoclave
160 degrees for 1 hour
duration and temperature for hot air sterilizer
Culture
grow and multiply in or on a culture medium
boiling
has more rapid action and more penetrating power compared to dry heat
Inoculation
implantation or introduction of specimen into the culture medium
pink color turbidity gas production
indications of bacterial growth
turbidity change in color gas bubbles
indications of growth in broth
bacteria have multiplied will cling together to form a visible mass called a colony
indications of growth on agar
protein coagulation alreration of protein structure cell membrane damage
mechanisms of action (chemical agents)
classic pasteurization (62 degrees for 30 minutes)
method of destroy disease producing organisms without damaging other important compounds such as viamins or nutrients in milk
streak plate
most practical and most useful method of obtaining discrete/isolated colonies and pure cultures
Inoculum
organism or specimen introduced into culture medium
dry heat ( hot air)
other methods which use the same principle include direct flaming of hoops and incineration of biological waste
Cultivation
process of growing microorganisms by taking bacteria from the infection site by some means of specimen collection and growing them in the artificial environment of the laboratory
autoclave
protein coagulation
boiling
protein coagulation
dry heat (hot air)
protein oxidation
solid (agar)
streak, stab, stab and streak in inoculum
liquid (broth)
suspend and mix in inoculum
hot air
use of ovens carbonization of organic material and spore destruction
pour plate or spread plate
used as means of determining the approximate number of viable organisms in a liquid like water or milk
Filtration
used for carbohydrates solutions and other liquids that
Inspissation
used for media containing serum or certain proteins
yellow
what color presence of alkaline?
indicates contamination of bacteria
what does it mean when bacterial growth changes from pink to yellow to pink?
2% sodium carbonate
what increases the killing effect of boiling?
air dry
what is done to preserce bacterial morphologies?
heat fixation
what is done to prevent washoff during staining?
single or pairs
what is the arrangement of E.coli?
