Micro Lab 3 and 4
Obtain too much culture will cause cells to pile on top of each other Use too much saline will increase drying time Use saline with liquid culture. Only broth is used with liquid culture. Adding saline will result in too little cells in the smear.
. Discuss three errors that may occur when preparing the smear.
Yes, if it is Myobacterium tuberculosis that is an acid fast bacteria that cause tuberculosis.
If a patient has acid fast bacteria in the sputum specimen they provided, does this mean they have tuberculosis
Purple, pink
In general, bacteria have one of two types of cell walls which determine the Gram stain result. When the Gram stain is completed, if the bacteria are ____, the bacteria are called Gram positive. If the color is_______, the bacteria are called Gram negative.
crystal violet
In the first step of the Gram stain procedure _____ ____ stain is applied and initially stains both Gram positive and Gram negative peptidoglycan layers a purple color
tryptophanase, tryptophan, indole pyruvate Kovac's red ring
Indole production in SIM Some bacteria produce the enzyme ________and thus are able to hydrolyze the amino acid tryptophan producing___ AND _____ Indole can be detected by the addition of Kovac's reagent. will have a_____ ____ on to
Klebsilla pneumonia
Klebsilla pneumoniae
Crystal violet; stain peptidoglycan of gram (+) and gram (-) purple Gram's iodine; use as mordant. cause Crystal violet to bind to gram(+) cell wall. Gram's Alcohol; decolorizing gram(-) and lock in iodine and crystal violet in gram(+) safranin; stains gram(-) pink-red Purified water; use to wash each reagent between each step
List the Gram stain reagents and the purpose of each in the Gram stain procedure
TRIPLE SUGAR IRON test
Media designed to differentiate bacteria based on glucose fermentation, lactose fermentation, sucrose fermentation, and sulfur reduction
citric acid utilization citrate
Simmon's Citrate Agar SCA is used to differentiate gram negative bacteria on the basis of____ ___ ______ . The agar medium contains sodium ____as the only available carbon source, and a single nitrogen source as well. If bacteria grow, it means they used the citrate as their carbon source.
ammonia blue green
Simmon's Citrate test Organisms which have the enzyme citrase, are able to utilize the sodium citrate and nitrogen sources and produce ______, which converts the agar to an alkaline pH and a ___color. If they cannot use these carbon and nitrogen sources, the medium remains ___
green = negative blue = positive
Simmon's citrate test
glycocalyx , capsule
Some bacteria are surrounded by a ____which consist mostly of polysaccharides and some polypeptides. Slime layers are a type of glycocalyx that are loosely bound. If the glycocalyx is well organized and tightly bound, it is referred to as a ___
Lipopolysaccharide
This difference in structure explains how the dyes interact with these different cell walls. gram negative stains pink because of the _____________.
teichoic acid
This difference in structure explains how the dyes interact with these different cell walls. gram positive stains purple because of the _____________.
ammonia carbon dioxide pink
UREA HYDROLYSIS: Some bacteria have the urease enzyme which hydrolyzes certain amino acids and urea to ____and ____ _____. When ammonia is produced the pH becomes ______(basic) Urea agar contains urea, peptones, glucose and phenol red. When urease positive bacteria are inoculated, the medium becomes alkaline turning the phenol red indicator bright ____.
Identification of unknown bacteria in relatively short time
What clinical information does a Gram stain provide?
Mycobacterium and some Nocardia
What genus of bacteria is typically acid fast?
Allow the bacteria to breakdown large molecules into smaller ones. The smaller molecules then, can enter the bacteria cell and be used as nutrient
What is a bacterial exoenzyme and what is the function? Why are they important for bacteria
mycolic acid in the cell wall
What is the cellular target of the acid fast stain?
The function of spores is to protect the DNA from radiation, extreme temperature, harsh chemicals, etc
What is the function of spores? W
to stick the bacteria to the slide so it will not be washed off during staining
What is the purpose of heat fixation of a smear?
To obtain accurate staining results
What is the purpose of the smear preparation?
To show existence of chemical reactions by changing their color. urea: phenol red SCA: bromthylmol
Why are indicators used in the differential media tubes? Name the indicators in the urea agar tube and the SCA tube.
old cultures may yield in false positive/negative results
Why does culture age affect the results of a Gram stain?
So we can begin treatment for infection early.
Why is rapid identification important in a clinical setting?
To obtain accurate staining results
Why is rapid identification important in a clinical setting?
Because it is showing different characteristics of different types of bacteria.
Why is the media in the test tubes referred to as differential media?
Tryptophanase released by the bacteria →hydrolyze amino acid tryptophan→produce indole & pyruvate
Write the chemical reaction for indole production.
Bacillus ,
___and ____are among bacteria that commonly produce endospores when their environmental conditions become unfavorable, most notably due to lack of nutrients.
Endospores
___are structures that form inside some bacteria. They are a dormant form that survives harsh environmental conditions.
mordant
Gram's iodine is added. It is called a _____and forms an insoluble chemical complex with crystal violet
sulfur H2S sulfate black
H2S production in SIM ____reduction. produces _____. The SIM media contains ferrous ____(iron salts) that will turn ____when they combine with H2S gas
Opportunists Pathogens gram negative because some of them can cause diseases in humans and animals.
Describe 3 properties of Enterobacteriaceae. Why is it important to identify them correctly?
Enterobacteriaceae
Differential media will aid in the identification of unknown gram negative rods in the __________ family
Preforming the decolorizing step incorrectly or using too thick or old culture may result in false negative or positive results
Discuss two causes each for false positive and false negative stain reactions
Tuberculosis,Leprosy
Discuss two diseases caused by acid fast bacteria.
Yes
Do animals get tuberculosis? Discuss this with your instructor.
TSI, SIM, SCA, UREA
4 standard differential tube media
germination cycle
Autoclaves, sterilizing gases, liquids, and ionizing radiation are some of the methods used in hospitals to insure that spores are killed
biofilm
Biofilm community of bacteria that stack to a surface. Biofilm make the bacteria resistant to antibiotics and increased virulence.
Urea agar
Contains peptone, glucose, urea, and phenol red. During incubation, bacteria possesing urease will produce ammonia, which raises the pH of the medium, turning the indicator fucshia pink.
Enterobacteriaceae Family
Enterics Large family of small, non-spore-forming Gram-negative rods Many members inhabit soil, water, decaying matter, and are common occupants of large bowel of animals including humans. Most frequent cause of diarrhea through enterotoxins Enterics, along with Pseudomonas sp., account for almost 50% of nosocomial infections.
gastrointestinal microbiota, opportunists, and pathogens.
Enterobacteriaceae Family includes. 3 These bacteria cause diarrhea, dysentery and hemolytic uremic syndrome.
differential
Enzymatic activity can be observed by using _________media which often changes appearance, for example, color, when an enzyme causes the pH to change (becomes acidic or basic).
Cultural isolation & identification skin testing
Explain a screening test and a more definitive test for diagnosis of tuberculosis.
Crystal violet absorbed in the peptidoglycan layer (cell wall) of gram(+) & gram(-) dye them purple. Gram's iodine prevent crystal violet to be absorbed in the cell membrane. Gram's alcohol dissolve and can remove the crystal violet/iodine but also shrinks the pores of the peptidoglycan. because gram(+) cell wall is thicker than gram(-) cell wall some of the crystal violet/iodine got locked in deep in gram(+) cell wall while the gram(-) turn colorless. Safranin stains gram(-) pink. because the dark purple color of gram(+) is overpower the pink color of safranin, gram(+) stays purple
Explain how bacterial cell wall structures account for the two different Gram reactions
When environmental conditions become unlivable for some bacteria, they duplicate their chromosome and surrounded by a very thick cortex of peptidoglycan and spore coat
Explain the cycle in some bacteria of making spores and germinating them.
Fermentation of any of the the three sugars, glucose, lactose, and fructose by the bacteria. Peptoniziation: Usage of peptones (amino acids in the media) as an additional food source by the bacteria. Production of H2S
Explain the different types of metabolic processes that can be observed and read from the triple sugar iron agar media
Explain the meaning of tuberculosis as a re-emerging infection
Explain the meaning of tuberculosis as a re-emerging infection
Because their presence on the bacteria cells increase the chances for serious disease.
Explain why capsules are considered virulence factors?
because it allow us to tell the difference between two types of bacteria Gram(+) & Gram(-)
Explain why the Gram stain is a differential stain.
Test: Ability to move away from Stab site Positive: Turbidity, spread of growth
Motility in SIM:
Mycobacterium
Mycolic acid is a waxy material that is found in the genus _____
Proteus vulgaris E. coli
Name a possible pathogen that is motile and produces indole.
Streptococcus pneumonia
Name an encapsulated bacterial pathogen that is a common cause of pneumonia
Two streptococcus species form biofilm on our teeth (plaque). S. mutans & S. sanguis
Name two genera of bacteria that produce endospores. Name an important pathogen in each genera
Chromagar
One new media is _____orientation which helps differentiate many of the members of the Enterobacteriacea family rapidly based on the color of the colony. This media is often used for rapid differentiation of Gram negative bacteria in urinary tract infections.
E. coli and Proteus.
Other Enterobacteriaceae members are well known causes of urinary tract infection, such as _____ and ____
decolorization
Removal of color with alcohol is called _____
Simmon's Citrate Agar
SCA MEDIA
Sulfide-Indole-Motility
SIM stands for... This is a combined media that tests for motility, sulfur reduction to hydrogen sulfide (H2S), and indole production from the amino acid tryptophan.
true
Salmonella and Shigella and even some strains of E. coli are intestinal pathogens
bromthymol blue green blue
Simmon's Citrate Agar SCA also contains the indicator, ______ ____, which is ____at pH 6.9 and ___at pH 7.6 and above
phenol red yellow orange/pink
TRIPLE SUGAR IRON AGAR: The indicator, ____ ___, is added to monitor the pH. This indicator is ___in acidic (A) pH and ______/ ___in alkaline (K) pH. medium contains ferrous sulfate to detect sulfur reduction and H2S production.
fermentation sucrose lactose
TRIPLE SUGAR IRON AGAR: This medium contains three carbohydrates and differentiates bacteria on _____of glucose, ___, and ___, or not
slant: Yellow (A) acidic, Orange/pink (K) alkaline Gas: bubbles or breaks H2S: black =+
TRIPLE SUGAR IRON test. slant butt : always acidic(A) Gas: H2S
Triple Sugar Iron
TSI stands for
differential , mycolic acids
The Acid fast stain is a ______stain that detects the presence or absence of ____ in the bacterial cell wall.
differential
The Gram stain is a ________stain which uses various dyes to differentiate two large groups of bacteria based on their cell walls (peptidoglycan layer).
true
The acid fast stain is used as a presumptive test for the presence of acid fast bacteria in clinical specimens where infections with Mycobacterium are suspected. Mycobacterium tuberculosis causes tuberculosi
virulence factor
The mycolic acid is 50% of the dry weight of the Mycobacterium cells and is sometimes referred to as "cord factor" as it results in clumping and aggregating of the bacterial cells. It is a ____ ___, allowing survival of Mycobacterium in phagocytes, and contributing to resistance to disinfectants and antimicrobial therapy and may make a mycobacterial infection more difficult to treat
positive; negative
acid fast stain results Read pink rods as acid fast positive. Read blue cocci as acid fast negative.
Serratia, Enterobacter, Klebsiella
enterics cause pneumonia in immune compromised individuals such as ____, ______, and _____
Gram-negative bacteria
gram negative or positive?
Gram-positive bacteria
gram negative or positive?
Gram-positive bacteria
have a thick peptidoglycan layer (up to 25 layers),
gram negative
have a thin layer of peptidoglycan and an outer membrane
normal flora
many of the Enterobacteriaceae are ____ ___, when they are found in hospitals, they are often resistant to multiple antibiotics and cause nosocomial infections in both humans and animals.
GRAM STAIN PROCEDURE
smear, heat fix, stain with crystal violet 30 seconds, wash, apply grams iodine (1) minute, wash, apply 95%alcohol drop by drop, 5 - 10 seconds or until alcohol flows from the slide until very little or no color is coming off. Do this step carefully wash, counterstain safranin, 45 sec. wash, blot dry, examine.