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MacConkey is used as a selective media because only GRAM -NEGATIVE will grow
Culture A = Gram-positive
BIOD 171 LAB NOTEBOOK UNKNOWN PATHOGEN ANALYSIS PORTAGE LEARNING 2023
KS PL09 Unknown Pathogen Analysis
Objective: To identify an unknown pathogen(s) based on the given assays developed during this course.
Notes: Two samples from the hospital were brought to your lab for diagnosis. After culturing the samples on
LB agar plates another co-worker began the analysis. However, due to an emergency your co-worker has left
the remaining work to you. Before leaving your co-worker informs you the 2 liquid cultures were accidently
combined before the Gram stain was started. However, the 2 cultures remain separated on LB plates for future
tests.
Goals: To correctly identify the 2 pathogens
Results: Highlighted in yellow Gram-negative rods
Gram-positive clusters
Gram Positive bacteria = PURPLE o Thick peptidoglycan layer (outside layer of bacteria) which retains the crystal violet stain
Gram Negative bacteria = PINK o Thin (single) peptidoglycan layer is damaged by the alcohol rinse step and the crystal violet stain is washed away o Pink color is derived from Safranin which is the secondary counter stain
Blood agar plate- gets its name from the red color and that it contains blood cells which will be important as a nutrient
source for the plate. There is actually a derivative of blood agar plates, that if you remove the RBC’s, you then get TSAYE (these are your tryptic soy agar yeast extract). That is a foundational component of blood agar plates.
Culture A = Has a zone of clearing
Non-selective media: Important for the expansion of unknown bacteria Basically, anything will grow on the media plates; it provides the carbon source & nutrients to help enhance and encourage the bacteria to grow
Culture B was streaked with the 4 - phase method and has no zone of clearing
EMB (eosin methylene blue)- a selective media that again, grows for Gram-negative bacteria. While it looks dark red, when it is tilted you can see a bluish/green sheen to it.
Can also be used as a differential media
You can differentiate different subgroups of Gram- negative bacteria.
If you have a strong lactose fermentation ability, the colonies will actually turn green on the plate
However, if you only have partial ability to ferment lactose, the colonies will turn pink
If the bacteria do not possess any enzyme that are capable of breaking down lactose, there will be NO color change in the bacteria (just the color that grows on the plate)
Culture A = Since no growth is noted; this would be Gram- positive; incapable of breaking down lactose
Culture B = Gram-negative; strong lactose fermentation ability
Staph
Aureus
E. Coli
Coagulase negative = no precipitant Coagulase POSTIVE = PRESENCE OF FIBRIN AGGREGATES (CLOTS) Qualitative test to determine the presence or absences of coagulase, an enzyme that plays a role in the formation of blood clots. (Coagulase binds to CRF [reacting factor] together they interact with fibrogen to form fibrin {CLOTS} so if a bacteria possesses coagulase, it will be coagulase positive and you will see precipitate in the bottom of tube. Coagulase positive is important in bacteria because it’s used as a defense mechanism. It can be used against antibody recognition because antibodies are circulating through the bloodstream, they have a hardened fibrin coat of bacteria. It can actually prevent the bacteria or the antibodies from recognition. It can also help the bacteria escape from phagocytosis, which can lead to their own death. Also note that one of the coagulase positive bacteria most common is that it’s present in STAPH AUREUS, which has a resistance to ABX and the immune system antibodies
Culture A = Coagulase POSTIVE (clotting) Culture B = Coagulase negative = no precipitant/clotting
