Micro Lab Practical 2
regarding bacterial families, what does an oxidase negative test result indicate? what does an oxidase positive test result indicate?
- -> Family Enterobacteriaceae (genera: Escherichia, Citrobacter, Enterobacter) + -> genera: Neisseria, Alcaligenes
Two different bacterial samples, A and B, were inoculated onto Blood agar. What conclusions can you make?
- Specimen A exhibits beta hemolysis - Specimen B exhibits gamma hemolysis
Explain the function of peptones in the broth media
- alternative substrate for nonfermenting organisms - fuschia - energy source
K/A
- fermentation of glucose only - H2S gas not produced - red slant/ yellow butt
A/A +H2S
- fermentation of glucose only - H2S gas produced - yellow slant/ black precipitate in butt
A/A
- fermentation of glucose, and lactose and/ or sucrose - H2S gas not produced - yellow slant/ yellow butt
define/ describe a coliform
- gram negative - oxidase negative - enteric bacteria - ferment lactose w the production of acid and gas when incubated at 37 degrees Celsius - members of the family Enterobacteriaceae
Klebsiella pneumoniae on MacConkey agar
- growth - gram-negative enteric bacterium - colonies are yellow w red edges - lactose fermenter - has capsule - coliform
what are two pathogenic organisms that are positive for urease production? what diseases do they cause?
Helicobacter pylori - gastric ulcer disease Proteus vulgaris- UTIs
Streptococcus pyogenes
Lancefield Group A causes strept throat
describe the biochemical mechanism that differentiates the coagulase positive bacteria from the coagulase negative bacteria
For coagulase positive bacteria-> fibrinogen reacts w coagulase to become fibrin that does not dissolve in H2O leading to the formation of a fibrin clot
In the urease test, a red coloration of the broth after 24 hours of incubation is considered __________.
a negative result due to no urease activity
2,3-butanediol fermentation is detected by the presence of which organic molecule
acetoin
if an organism uses the mixed acid fermentation pathway to break down glucose, the final pH in the medium will be __________, which can be detected by the pH indicator ________________
acidic, methyl red
what hemolytic pattern is seen with Streptococcus pneumoniae? what test separates this organism from other streptococci with the same hemolytic reaction
alpha hemolytic optochin susceptibility test (sensitive to low concentrations of the chemical optochin)
superoxide radical
toxic byproduct produced by reactions in the election transport chain
The only carbohydrate in a phenol red lactose broth tube is the sugar lactose.
true
The indole production test determines the ability of microorganisms to __________.
degrade tryptophan
define differential media. what is the ingredient that makes MSA a differential medium
differentiates between 2 groups of bacterial mannitol makes the MSA plate a differential medium
A bacterial species may produce H2S but not turn the butt yellow.
false
glycolysis
glucose catabolism; common first step in both cellular respiration and fermentation
what does the salt select for
halophiles (salt loving)
Virulence factors are structures or molecules that allow pathogens to successfully invade and colonize a host. what are hemolysins considered virulence factors
hemolysins are toxins produced by certain bacterial organisms that destroy RBCs and damage human tissue (can now take their iron)
Catalase breaks down which compound into water and oxygen gas?
hydrogen peroxide
if you removed the ferrous sulfate from the tube, what would you no longer be able to detect
hydrogen sulfide
mixed acid fermentation is detected by the presence of which organic molecule
methyl red, which detects for long-term stable acid products (min incubation time of 72 hours) + result = red - result = no color change
what is the catalase test for all streptococci
negative
gamma hemolysis
no hemolysis
Streptococcus pneumoniae
non-group causes pneumonia
why is alpha hemolysis also called partial hemolysis
partial breakdown of hemoglobin in RBC (still some material that will turn it a greenish color)
list the 5 characteristics of family Enterobacteriacea
1) They are small, Gram-negative bacilli and some of them are coccobacilli 2) they are all oxidase negative 3) they are all facultative anaerobes 4) they are all saccharolytic, capable of fermenting glucose into lactic acid or other acidic compounds 5) most of them are capable of reducing nitrate to nitrite
what are two mistakes that a microbiology student might make that could lead to a false positive result for the oxidase test? in your answer, briefly explain why both of these mistakes result in a color change
1) one issue could be if the test is not read fast enough, needs to be read within 30 seconds or less (if a color change is not observed within 30 seconds, the test is negative) 2) metal loops can not be used with oxidase test (the loops contain iron and with repeated heat-sterilization, oxidation products can build up on the loop. these products could react with the reagent, yielding a false positive)
Lab Review
22- Coagulase Test
Lab Review
23 - Hemolytic Patters: Blood Agar
Lab Review
24 - Identification of the pathogenic streptococci: bile esculin agar, 6.5% NaCl broth, and bacitracin and optochin susceptibilty tests
Lab Review
25 - oxidase test
Lab Review
26 - MacConkey Agar
Lab Review
27 - Triple Sugar Iron Agar
Lab Review
28 - Urease test
are oxidase negative bacteria capable of still performing aerobic respiration? Explain
A negative result for the oxidase test does not imply that the bacteria are not capable of aerobic respiration, a negative simply means that the organism doesn't have cytochrome c oxidase specifically as part of its electron transport chain, but it could have another cytochrome complex instead
When TSIA is inoculated with Salmonella typhimurium, thiosulfate is reduced to H2S. Explain
A/A + H2S - sulfur reduction occurred causing the butt to turn black - glucose, lactose and /or sucrose fermentation
Biochemistry of oxidase test
Before reaction: colorless - Phenylenediamine (reduced) + cyt c oxidase (oxidized) -> reagent added to bacteria -> After reaction: purple - Phenylenediamine (oxidized) + cyt c oxidase (reduced)
describe how you can differentiate between S. aureus and S. epidermidis growing on MSA
Both of the bacteria will grow on MSA plate. S. aureus is able to ferment mannitol and turn the plate yellow while S. epidermidis can not and the plate remains pink.
Urease is the enzyme that breaks down urea into what compounds?
CO2, H2O, and NH3
You observe colonies growing on EMB agar that are blue-black with a metallic green sheen. What species of bacteria is this likely to be?
E. coli
Why are the coliform plates incubated at 37° C?
They grow best at body temperature.
Can Group A and Group B Streptococci be differentiated from each other by observing the results of their Gram stains and hemolytic reactions on blood agar? explain. if not, what test can differentiate between them?
No, because they are both gram + and have beta hemolytic reactions. The bacitracin test would be able to differentiate between the two.
Klebsiella is capable of fermenting glucose, lactose, and sucrose. if you inoculated this organism on TSIA but forgot to check it after 24 hours, what would you expect to see after 96 hours of incubation if you left the media in the incubator over the weekend?
Reversion, because the bacteria would have moved on to eating proteins bc they ate all the sugars, now give a result of K/K (false negative)
In lab you inoculate a plate of MSA with a mixture of S. epidermidis and Micrococcus luteus. after incubating the plate for 24 hours at 37 degrees Celsius, describe what you would observe and how you would differentiate between the two.
S. epidermidis = growth Micrococcus luteus = no growth
what two streptococci have capsules
S. salivarius and S. pneumoniae
Enriched blood agar permits the demonstration of the hemolytic properties of what group of microorganisms?
Streptococci
Streptococcus mutans
Viridans group causes tooth decay
what are the ingredients that make MacConkey agar a selective medium? what do they select for?
bile salts- inhibit any non-enteric Gram-negative bacteria crystal violet- inhibit growth of most Gram-positive bacteria select for gram negative enterics!!
organisms that have citrate permease and citrase are able to use citrate as their only source of __________
carbon
why is beta hemolysis also called complete hemolysis
complete destruction of RBC (clear)
explain the role of phenol red in the urea medium. how does phenol red help in the detection of change in the medium
phenol red- pH indicator @ neutral pH = pale orange, yellow (urea is not hydrolyzed) @ basic pH = hot pink = + result (organism produces urease and can hydrolyze urea as a nutrient source, ammonia accumulates) a peach color = slow urea hydrolysis
A specimen was inoculated onto the Triple Sugar Iron (TSI) medium pictured here.
Hydrogen sulfide (H2S) was produced. The bacterium is able to ferment glucose. The pH of the agar decreased following incubation.
A simple test to differentiate between Staphylococcus and Streptococcus is the addition of H2O2 to a colony to see if bubbles form, which are positive for catalase. Which organism would be positive?
Staphylococcus
could you have results of this test that are positive for motility but negative for utilizing the electron transport chain? Explain your reasoning and describe how the growth in the culture tube would appear
Yes, Fermentation and lack of the red color
Would a bacterial species be considered urease positive if you have to incubate it for 48 hours to get the pink positive reaction?
Yes, this increased incubation means that the bacteria express a low level of the enzyme or their enzyme has a low level of activity
Why must you perform multiple dilutions for each food type?
You do not know the initial concentration of bacteria in the food
If you inoculated a triple iron-sugar slant with Shigella, a glucose fermenter, your results should be what?
a red slant with a yellow butt
A bacterial species that is a gas producer and has the ability to ferment lactose is added to a Durham tube of phenol red lactose broth. What will the results be the next day?
a yellow broth due to acidification and color change of the phenol red, and a gas bubble will be trapped in the inverted tube
Is nitrification an aerobic or anaerobic process?
aerobic
You inoculate an organism onto blood agar and observe a clear zone surrounding the bacterial growth. What type of hemolysis is indicated by this observation?
beta hemolysis
Explain the function of the Durham Tube in the broth media
collect gases produced by bacteria
Which of the following is indicative of the presence of E. coli?
colonies on EMB agar with a metallic sheen
catalase test
differentiates the Gram-Positive bacteria
Sulfanilic acid and α-naphthylamine are red reagents that become clear in the presence of nitrite.
false
reduction
the gaining of electrons
oxidation
the loss of electrons
2 ATP
the net amount of energy produced in fermentation
pyruvic acid
the organic molecule that acts as the election acceptor in fermentation
why is it advantageous for enteric bacteria to perform anaerobic respiration using nitrate reduction
there is no O2 in digestive system
A urease-negative bacterial species is allowed to grow in a urea broth overnight. What color would the broth be the next day?
yellow or orange, due to the acidification of the broth
In the nitrate reduction test, only after the addition of zinc to your 24-hour incubated solutions A and B is there a color change. What does this indicate?
No nitrate was reduced by the bacteria.
what can you conclude about the Acid-Fast stained specimen?
- The pictured bacteria are acid-fast - The pictured bacteria have mycolic acid in their cell walls - The pictured bacteria are possibly a type of mycobacteria (HINT: This image is of Acid-Fast bacteria. The waxy mycolic acid in the cell wall of these bacteria causes them to clump together in a formation known as cords)
motility agar differentiates bacteria on what two properties
- motility - cellular respiration
You observe a yellow zone surrounding growth on mannitol salt agar. What does this indicate about the organism?
The organism is able to ferment mannitol.
Name the three steps of cellular respiration
1. Glycolysis 2. Krebs Cycle 3. Electron Transport Chain
Lab Review
17- Phenol Red Broth
Lab Review
18 - Nitrate Reduction
Lab Review
19 - Motility Agar
Which of the following bacterial concentrations would produce a TFTC plate at the 10-4 dilution?
2 × 10^5 organisms/ml
Lab Review
20 - Catalase Test
Lab Review
21 - Mannitol Salt Agar
How long does nitrate broth need to be incubated for after being inoculated?
48 hours
You found 67 colonies on the ground beef plate with the 10-2 dilution. What is the concentration of bacteria per milliliter of beef?
6.7 × 10^3 CFU/ml
If you had fresh lettuce containing 9.3 × 103 organisms/ml, how many colonies would you expect to see on a plate that represents a 10-2 dilution?
93
After running this experiment, you find that the carrots you tested had only 15 bacteria/ml. Your lab partner states that this number is so low that the carrots do not need further treatment. Why should you disagree?
A low number of organisms does not indicate the absence of pathogens
The methyl red test is based on the glucose utilization of Enterobacteriaceae, resulting in acidic end products. The coloration of the reagent is based on what further reactions?
A red coloration of the methyl red indicates that the bacteria are maintaining an acidic environment, while a yellow coloration indicates that this bacterial species converts these acidic products in other metabolic pathways.
Which of the following would produce a countable plate?
A solution containing 1.9 × 10^4 at a 10^-2 dilution
Write out the formula for aerobic respiration
C6H1206 + 602 -> 6H2O + 6CO2 + ENERGY (38 ATP)
describe the source of electrons that enter the electron transport chain
NADH FADH2
Two specimens, A and B, were inoculated into Urea broth. What can you conclude from your observations?
Bacterium A converted urea to ammonia Bacterium A increased the pH of the medium Bacterium B decreased the pH of the medium Bacterium B does not produce urease
Which of the following describes an INCORRECT association regarding hemolytic activity?
Beta hemolysis; results in a greenish halo around the bacterial growth
Write out the equation for the catalase reaction
H2O2 (substrate) -> catalase (enzyme) -> H2O + O2 (products)
What creates the black precipitate that is found in the bottom of a TSI agar slant inoculated with Salmonella?
H2S production
What is the purpose of adding salt to mannitol salt agar?
High concentrations of salt inhibit the growth of non-halophilic organisms but allow for the growth of bacteria from the genus Staphylococci.
After 24 hours of incubating your bacterial isolate to test for carbohydrate fermentation capabilities, your Durham tube shows a yellow broth with a trapped bubble. What does this indicate?
The bacteria were able to ferment the sugar source and produce CO2
how would you interpret you results if you inoculated a carbohydrate broth tube and after incubating the tube for several days, the broth had turned hot pink in color ?
The broth was incubated past 24 hours and experienced reversion because the carbohydrate source ran out of supply and the bacteria began to digest the peptone. This increased the pH and the color went from yellow to hot pink.
You observe red colored colonies growing on MacConkey agar. What does this indicate about the organism?
The organism is able to ferment lactose.
An unknown specimen was grown on Mannitol Salt agar (MSA). Observe the pictured result and answer the following question.
The organism tested ferments mannitol. The organism tested is able to grow in the presence of high salt. The organism tested is possibly a type of Staphylococci. The media used in this test is selective. The media used in this test is differential. The tested organism is likely to be coagulase positive. The media used in this test is good for growing Staphylococci.
briefly explain how you would know if the bacteria growing in the phenol red broth are producing energy by cellular respiration or fermentation
There will be a color change yellow = fermentation
from a biochemical standpoint, why can anaerobic organisms not survive in the presence of oxygen? what enzyme are they lacking?
They cannot survive because they can not handle free radicals They either lack superoxide dismutase, peroxidase, or catalase
An unknown specimen was grown on Indole media. Based on your observation of this specimen choose the appropriate statements.
This test is a part of the IMViC panel of tests This media is inoculated by stabbing the agar in the tube. This is a picture of a negative test result.
Results of motility test
Tube 1: strong positive for motility; positive for respiration Tube 2: positive for motility; positive for respiration Tube 3: negative for motility; positive for respiration Tube 4: inconclusive for motility; positive for respiration
A Lactose Fermentation test was performed on two different bacterial samples, A and B. Based on your observations what can you conclude?
Test organism A ferments lactose Test organism A produced gas Test organism A lowered the pH of the medium Test organism B does not ferment lactose
Lab Review
29 - IMViC
Escherichia coli on MacConkey agar
- growth - gram-negative enteric bacterium - red colonies - lactose fermenter - coliform
Salmonella typhimurium on MacConkey agar
- growth - gram-negative enteric bacterium - yellow colonies - not a lactose fermenter
The presence of the enzyme tryptophanase is indirectly detected by the waste product _______________ reacting with ________________ reagent.
- indole - Indole Kovac's reagent
K/K (-/-)
- no carbohydrates fermented - H2S gas not produced - red slant/ red butt or no change
Staphylococcus aureus on MacConkey agar
- no growth - not a gram-negative enteric bacterium - no colonies - not a lactose fermenter - not an enteric pathogen
Explain the function of phenol red in the broth media
- pH indicator - fermentation of carbs -> acid pH < 6.9 (yellow) - enzymes hydrolyze peptones -> basic pH > 8.2 (fuchsia)
Explain the function of carbohydrates in the broth media
- substrates for fermentation - yellow - nutrition, energy source
Below is a Glucose Fermentation test. Choose all appropriate statements about the Glucose Fermentation test.
-The mouth of the tube should be flamed prior to inoculation and immediately before capping the tube -This media is inoculated using an inoculating loop -This test detects a fermentation reaction -The test contains phenol red which turns yellow under acidic conditions
Red precipitate observed after reagents A & B and zinc were added
1) negative for anaerobic respiration 2) negative for nitrate reduction only 3) negative for denitrification
clear broth observed after reagents A & B and zinc added
1) positive for anaerobic respiration 2) negative for nitrate reduction only 3) positive for denitrification
red precipitate observed after reagents A & B only added
1) positive for anaerobic respiration 2) positive for nitrate reduction only 3) negative for denitrification
what genus is not inhibited and can grow in 6.5% salt broth
Entercoccus
Streptococcus agalactiae
Lancefield Group B causes neonatal sepsis
Enterococcus faecalis
Lancefield Group D causes UTIs and VRE infections
Two different bacterial samples, A and B, were inoculated onto Simmons Citrate agar. What conclusions can you make?
Specimen A can use sodium citrate as a carbon source Specimen A raised the pH of the medium as a result of its metabolic processes Specimen B cannot use sodium citrate as a carbon source
Two different bacterial samples, A and B, were tested with an Oxidase dry slide. This is a picture of the test results
Specimen A does not produce cytochrome oxidase. Specimen B produces cytochrome oxidase Specimen A shows a negative result Specimen B shows a positive result Specimen B most likely has a respiratory chain that uses oxygen as a final electron acceptor.
Two different bacterial samples, A and B, were inoculated into MR-VP (Methyl Red-Voges Proskauer) broth. The following image shows what the tubes looked like for the Methyl Red portion of the test.
Specimen A fermented glucose and formed low levels of acid end-products Specimen B fermented glucose and formed high levels of acid end-products Specimen B can likely convert glucose to lactate, formate, or acetate
Two different bacterial samples, A and B, were inoculated onto MacConkey agar. What can you conclude based on your observations?
Specimen A is able to ferment lactose Specimen A is most likely Gram-negative Specimen B is most likely Gram-negative
Two different bacterial samples, A and B, were analyzed with the Voges-Poskauer (VP) test. The results are pictured here
Specimen B fermented glucose and formed acetoin (acetylmethylcarbinol). Specimen B is fermented glucose and formed neutral end-products. Specimen B had a positive result for the VP test.
why is it important to stab the agar when inoculating blood agar plates
checks for the presence of oxygen-labile hemolysins
+G
cracks or lifting of agar gas produced during fermentation of carbohydrates
Explain why the coagulase enzyme is a virulence factor for those bacteria that can synthesize it
creates physical barrier that protects it form immune system
oxidase negative bacteria lack a specific protein known as
cytochrome c oxidase
what is the ingredient in MacConkey agar that makes this media differential? what two groups can be differentiated by this ingredient?
differentiation is achieved with the carbohydrate lactose that is supplied in the media alone with the pH indicator neutral red coliforms and nonfermenters can be differentiated by this coliforms -> red, acidic pH nonfermenters -> colorless, basic pH
identify and briefly describe all bacterial cell structures that allow bacteria to move independently
flagella pilli endoflagella
IMViC is an acronym for what tests?
indole, methyl red, Voges-Proskauer, and citrate utilization
when observing the results of the motility test, why do strict aerobic bacteria sometimes show inconclusive results
it is difficult to tell whether the bacteria can't move down tube from lack of O2 or lack of motility
what would be observed if reagent A & B were then added to this uninoculated tube
it will turn red (nitrate -> nitrite)
what is the ingredient that makes MSA a selective medium
salt
periplasmic space
location in bacterium where toxins are stored an destroyed by enzymes
fermentation
many microbes use this process as primary means of producing ATP without oxygen
organelles
membrane bound compartments of eukaryotic cells
How would the use of selective and differential media decrease the time necessary for identification of clinical specimens or environmental samples
multiple tests at the same time = less tests!
what is the catalase result for all streptococci
negative
the phenol red test in the lab detects fermentation by the pH changes in the medium. since bacteria can generate ATP by both cellular respiration and/ or fermentation, are the products of cellular respiration acidic, neutral, or basic?
neutral
the reagents added in Part 1 of the nitrate test only react with and detect
nitrate (NO3^-)
the nitrate test differentiates bacteria on their ability to produce the enzyme
nitrate reductase
Partial nitrate reduction results in the formation of __________
nitrite and water
what is indicated when the bacteria growing along the stab do not turn red
not going through cellular respiration
what would happen if powdered zinc was added to an uninoculated nitrate broth tube
nothing
is nitrification a reduction reaction or an oxidation reaction?
oxidation reaction ammonia (NH3) or ammonium (NH4^+) -> nitrite (NO2^-) -> nitrate (NO3^-)
NAD+
oxidized form of nicotinamide adenine dinucleotide
identify the role of each indicator in TSIA
phenol red- pH indicator (pink at 7.4 and higher, yellow at pH below 6.8, and orange between this range) ferrous ammonium sulfate- turns black in the presence of hydrogen sulfide (H2S)
what is the catalase result for all staphylococci
positive
Mastering Micro
practice
white blood cells
produce oxygen radicals to destroy pathogenic bacteria
NADH
reduced form of nicotinamide adenine dinucleotide
Identify the following for the bile esculin test substrate detectable product indicator lancefield group
substrate- esculin detectable product- esculetin indicator- ferric citrate Lancefield group- D
explain the biochemistry of the urease reaction by identifying the following: substrate? enzyme detected? detectable product produced?
substrate- urea enzyme detected- urease detectable product produced- ammonia
