C6: Detection Methods

tissues

performed using touch preparations

swabs

must be rolled back and forth across a dry, clean slide

single-drop smear

• turbid

• high numbers of organisms are expected

• specimen is thick

centrifuged smear

• used when a small number of organisms are expected

• often clear

layered smear

• used for low-volume specimens

• a drop is placed on a slide and allowed to dry, then another drop is placed on top of the dried smear

albumin

may be added to CSF to help cellular material adhere to the slide

mucolytic agents

may be added to extremely mucoid specimens

gram stain

places bacteria into one of the two groups (g+/g-)

crystal violet

primary stain, gram staining

Gram’s iodine

mordant, gram staining

Acetone-alcohol

decolorizer, gram staining

Safranin red

counterstain, gram staining

acid-fast staining

used primarily to detect Mycobacterium species

kinyoun and ziehl-neelsen

two methods of Fuchsin stain

kinyoun method

also known as the cold method; uses a higher concentration of detergent to facilitate staining

Ziehl-Neelsen method

hot method; uses heat to facilitate staining and recommended for pulmonary TB

carbolfuchsin

primary stain, fuchsin staining

acid-alcohol

decolorizer, fuchsin staining

methylene blue

counterstain, fuchsin staining

malachite green

alternative for methylene blue

red

positive color for acid-fast staining

negative color for acid-fast staining

blue

auramine-rhodamine

acid-fast organisms fluoresce yellow or orange under a fluorescent microscope

methylene blue

used to observe metachromatic granules in Corynebacterium diphtheriae

acridine orange

fluochrome dye that stains both g+ and g- organisms both living and dead; used for low bacterial yield

saline mount

used to determine the biologic activity of microorganisms

hanging-drop procedure

same purpose as a saline mount but with a deeper field of focus and without the distorting effects from the coverslip’s weight; used to observe motility

darkfield examination

used to visualize organisms that are invisible by brightfield microscopy with great difficulty; demonstrates spirochetes

• 0

• +1

• +2

• +1

give me the grades for the following (neutrophil edition)

• <10

• 10-25

• >25

• presence of mucus

• -1

• -2

give me the grades for the following (eosinophil edition):

• 10-25

• >25

• liquid

• semi-solid

• solid

three kinds of culture media based on consistency/physical state

0.5%-1.0%

How much agar is in a semi-solid culture media?

2%-3% agar

amount of agar in solid culture media

agar

polysaccharide extract of seaweed/algae

• synthetic

• non-synthetic

• cell culture

three kinds of culture media based on composition

plated media

dispensed in sterile petri dishes

tubed media

dispensed in sterile test tubes

non-selective media

supports most non-fastidious bacteria

selective media

support the growth of one type of bacteria and not the other; may contain inhibitory substances

differential media

allows the grouping of bacteria on the basis of different characteristics displayed on the medium; may be selective or non-selective

enriched media

growth enhancers have been added

enrichment broth (selective enrichment)

designed to encourage the growth of small numbers of a particular organism while suppressing the other flora present in the specimen

transport media

prevents replication while keeping bacterial cells viable

blood agar plate

• most commonly used media

• for the cultivation of fastidious bacteria and demonstration of hemolytic reactions

chocolate agar

• dark brown in color

• blood agar except the RBCs are already lysed

• for the cultivation of Haemophilus and Neisseria

Thayer-Martin agar

• modified chocolate agar

• selective for Neisseria gonorrhoeae and Neisseria meningitidis

• antibiotics are added

• can be modified to have trimethoprim

Proteus spp.

What does trimethoprim inhibit?

Martin Lewis Agar

has vancomycin, colistin, and anisomycin (to inhibit yeast)

eosin methylene blue agar

• used for the isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli

• inhibits g+ bacteria

Mac Conkey agar

• same use as EMB

• contains bile salts and crystal violet to inhibit g+ bacteria

• neutral red functions as an inhibitor

Mannitol Salt agar

• for the selective isolation of coagulase positive staphylococci

• 7.5% NaCl inhibits most bacteria

• phenol red is the indicator used

phenyl ethyl alcohol agar

• selective for the isolation of gram+ cocci

• inhibits g- bacteria

• has blood

selenite broth (Sel-F)

• selective for Salmonella and some strains of Shigella

• should not be autoclaved

• toxic for most Enterobacteriaceae

thioglycolate broth

• supports the growth of aerobes, microaerophiles, and anaerobes

• contains Resazurin which functions as an oxidation-reduction indicator

• no more than twice autoclaved

• if green color has spread it must not be used

xylose lysine desoxycholate (XLD) agar

• for isolation and differentiation of Salmonella and Shigella from other g- enteric bacteria

• should not be autoclaved

• high NaCl content inhibits most enterobacteriae and g+ bacteria

• contains ferric ammonium citrate to visualize H2S production

• phenol red as pH indicator

hektoen enteric agar

• selective and differential of Salmonella and Shigella

• similar utility as that of XLD

brain heart infusion

• supportive medium for a wide variety of organisms

• general purpose medium

lowenstein-jensen medium

• selective enrichment for Mycobacterium spp.

• malachite green inhibits growth of other bacteria

• contains homogenized eggs for production of niacin

mueller-hinton

used for antimicrobial susceptibility testing

chromagar

provides a variety of colors for differentiation of specific isolates

• streaking

• pour plate

• spread plate

three kinds of inoculation techniques for isolation