Flash cards over lab procedures Blake
Mycobacterium are what
Once stained, they are resistant to what
Lipophilic
They are resistant to destaining due to the presence of cell wall-bound mycolic acids.
Carbol fuchsin penetrates the mycobacterial lipid with the aid of
Whereupon the organism resists destaining with
Phenol
Acid alcohol and consequently cannot stain with methylene blue stain.
Acid-fast stain procedure
1.) Prepare smear and heat fix
when using M.goronae only a brief heat fix is required compared with other mycobacterium that require a 2 hour heat fix in 60C.
2.) Flood slide with Kinyoun carbol fuchsin and let stain for 5mins.
3.) Wash with tap water
4.) Add acid alcohol and allow alcohol to flow over slide until slide appears colorless.
5.) Wash with tap water
6.) Add new methylene blue for 1 minute.
7.) Wash, blot, and examine under oil immersion.
+ number seen
1-2/300 fields
Minus number seen
0
1+
1-9 organism/ 100 fields
2+
9 or more/100 fields
3+
1-9/ field
4+
Greater than 9/Field
Auramine- Rhodamine Fluorochrome stain procedure
1.) Prepare slide and heat fix
2.) Flood fixed slide with Auramine Rhodamine T solution and let stain for 15 minutes
3.) Wash with tap water and flood with decolorizer for 2-3 minutes
4.) Wash with tap water and counterstain for 4-5 minutes
5.) Wash with tap water, blot gently and air dry
6.) Examine by fluorescence microscopy using a 40X objective for the detection of fluorescence organisms.
Auramine and rhodamine Penetration is aided by
Phenal
In the Auramine and Rhodamine method, the cellular components of the mycobacterium cell will have a
Golden yellow or orangish fluorescence.
What is the secondary stain used in the Auramine Rhodamine method?
Potassium permanganate is used to quench nonspecific background fluorescence
Decolorizer in Auramine Rhodamine method
HCL
Inoculate group B strep at what angle
90 degrees in CAMP tests
Arrow head hemolysis is positive result
MIC procedure using Macrobroth procedure
Note- the antibiotics that can be used on S. Aureus are Erythromycin or ampicillin, or other agents. The antibiotics used for Ecoil are levofloxacin or ampicillin.
1.) Label 10 disposable tubes 1-9 and growth control. Place 1 mL of sterile water in each tube. A 3 antibiotic disks to tubes 1 or 2. Allow to set at RT for 15 minutes.
2.) Add 1mL of water to tubes 2 and serially dilute tubes 3-9. Discard the last mL from tube 9.
4.) Prepare the inoculum of an organism to be tested by preparing a suspension of Tryptic soy broth equivalent to a 0.5 Mcfarland suspension.
5.) Make a 1:2 dilution of the Mcfarland suspension
6.) Add 1mL of diluted T-soy broth to each of tubes 1-9, and the growth control tube number 10 and incubate overnight at 35C
7.) On the following day, read tubes for turbidity. Read from tubes 1-9#, the MIC of the organism is the last tube showing no visible turbidity
8.) Calculate the MIC by determining the concentration of the drug in that tube and state it in the same units as the drug.
Micro broth Lab procedure
1.) Prepare a .5 Mcfarland standard with test organism in broth or saline
2.) Dilute
3.) Using a sterile pipet, add 0.1mL to each well of a microtiter plate
4.) cover and incubate for 18-24 hours
5.) read MIC
E-test susceptibility testing
1.)Prepare a 0.5 Mcfarland standard with test organism in broth
2.) Using a sterile swab, inoculate the entire surface of a Mueller Hinton agar plate
3.) Lay E test strip onto agar surface
4.) Incubate for 18-24 hours
5.) record MIC and interpretations
Preparation of Inoculum
1.)Select four to five colonies
2.) Transfer these colonies to a test tube containing about 5 milliliters of a suitable liquid medium, such as soybean casein digest broth, Trypticase soy broth.
3.) The inoculum density should be obtained by diluting it with sterile saline to obtain a turbidity equivalent to that of a freshly prepared turbidity standard 0.5 Macfarland.
MIC=MBC when
The plate shows no growth after incubation
The mordant is
Phenol
Platinum catalyst binds
Oxygen with hydrogen to produce H2O
