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6. Stains and Culture-1
Medical Technology (MD)
Our Lady of Fatima University
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BACTERIOLOGY LABORATORY
Our Lady of Fatima University College of Medical Laboratory Science- Valenzuela Campus Ma. Christy V. Gonzales, RMT, MPH
STAINS
- Simple stain
- Carbol fuchsin, methylene blue, gentian violet
- Differential stain
a. Gram stain
- For characterization of specimen a. Bartlett’s classification: sputum sample >10 epithelial cells/LPO and <25 PMN/HPO: saliva <10 epithelial cells/LPO and >25 PMN/HPO: sputum
Precautions Effects
Crystal violet rinsed too vigorously before it is complexed with iodine
Washed away the crystal violet and results to falsely gram (-)
Prolonged decolorization 2. Results to falsely gram (-)
Insufficient decolorization 3. Results to falsely gram (+)
Prolonged application of safranin
Results to falsely gram (-)
Insufficient application of safranin
Results to falsely gram (+)
Antibiotic-treated and dead or degenerating organisms
Gram variable/ atypical
Gram stain general rule:
All cocci are gram (+) EXCEPT:
All bacilli are gram (-) EXCEPT: Mycobacteria, Corynebacteria, Clostridia, Bacillus, lactobacillus, Listeria, Erysepilothrix, Nocadia, Actinomyces
All spiral organisms are reported as gram (-).
Yeast are gram (+).
b. Acid-fast stain - detection of mycobacteria in clinical specimen
Method Componen ts
Durati on
Test result
Report as
1.
Ziehl- neelsen’s method
- hot method
Carbol fuchsin (with heat) Acid- alcohol (3% HCl in 95% ethanol) Methylene blue/ Malachite green
5 mins 30 secs 30 secs- min
Red bacilli against blue backgrou nd
Blue bacilli against blue backgrou nd
Acid fast bacilli
Non-acid fast
2.
Kinyoun’s method
- cold method
Carbol fuchsin (no heat) Acid- alcohol (0% HCl in 95% ethanol)
3 mins 2 mins 30 secs
Red bacilli against blue backgrou nd
Blue bacilli
Acid fast bacilli
Non-acid fast
Methylene blue/ Malachite green
against blue backgrou nd 3. Pappenhei m’s method - differentiat e M. tuberculosi s from M. lacticola
Carbol fuchsin (with heat) Pappenhei m’s differentiat ing stain
Rosalic acid & Methylene blue in glycerine and absolute alcohol
2 mins 4- mins
Red/ pink bacilli
Colorless bacilli
M.
tuberculo sis
M. lacticola
4.
Baumgarte n’s method
Alcoholic carbol fuchsin 1% nitric acid in 95% alcohol
5 mins --
- Gabbet’s method
Alcoholic carbol fuchsin Acid- alcohol (3% HCl in 95% ethanol) Gabbet’s methylene blue solution
5 mins -- 20- secs
- Special stain
Method Components Test result
Structur e seen A. CAPSULAR STAINING
- Hiss staining method
Crystal violet/ Gentian violet - contr ast stain 20% copper sulfate solution
Clear/ halo around the bacteria l cell
Violet (same as backgro und)
Capsule
Bacteria
- Anthony’s method
1% copper sulfate solution 1% Alcian blue in 95% ethanol 1:1000 diluted carbol fuchsin
Unstain ed
Deep purple within the colorles s halo.
Capsule
Bacteria
The backgro und is colored purple. 3. Novelli’s method
Alcian blue 1:1000 diluted carbol fuchsin
Blue
Pink
Capsule
Bacteria B. METACHROMATI C GRANULES STAINING
- Loeffler’s alkaline methylene blue (L.A.M.) method
LAMB Dark blue
Blue
Polar ends of the bacteria
Bacteria 2. Albert method Albert’s stain Gram’s iodine
Blue to black
Blue to blue green
Green
Granules
Bands
Cytoplas m
C
STAINING
- Acetic acid method
5% acetic acid Carbol fuchsin LAMB
Red/ Pink
Blue
Spore
Bacteria
- Wirtz-Conklin method
5% Malachite green 0% Safranin
Green spherul es
Red
Spore
Bacteria
- Dorner method
Carbol fuchsin 10% Nigrosin
Red
Colorles s against a dark gray backgro und
Spore
Bacterial cell
D. CELL WALL
STAINING
- Dyar method Cetyl pyridium chloride Saturated solution of Congo red Methylene blue
Red
Colorles s
Cell wall
Bacteria
E. FLAGELLAR
STAINING
- Leifson’s method
Formalin Leifson’s flagellar stain
Red/ Purple
Colorles s
Flagella
Bacteria
- Fisher and Conn’s method
Carbol fuchsin Red
Colorles s
Flagella
Bacteria
F. LIPID
GRANULES
STAINING
Sudan Black B stain Xylol 0% safranin
Blue- black/ Dark bluish-
Fat globules
Cells
gray/ Black
Red G. NUCLEAR BODY STAINING
1% mercuric chloride 1% crystal violet (pH 6-8) 2-5% Nigrosin (pH 3-4) H. POLAR STAIN
1’s stain
Solution A Basic fuchsin- 0 g (90% dye) Methylene blue- 0 g (90% dye) Ethyl alcohol- 20 mL (95%)
Solution B 5% Phenol- 200 mL
Purple Polar bodies
CULTURE MEDIA
on or in which bacteria are grown
Bacterial cell multiply to sufficient numbers to allow visualization - Fastidious - With complex requirements: - Nonfastidious - Basic requirements
Cultivation
process of growing microorganisms in culture by taking bacteria from the infection site (in vivo) and growing them in the artificial environment of the laboratory (in vitro)
requires that the nutritional and environmental growth requirements
Classifications:
I. Phases a. Liquid/ Broth - nutrients are dissolved in water - bacterial growth (10 6 bacteria per mL) is indicated by change in broth’s appearance from clear to turbid b. Semi-solid - 0-1% agar **agarose: most common solidifying agent : melts at ≥95°C : re-solidify at < 50°C - used to observe motility c. Solid/agar - 2-3% agar d. Biphasic medium - both liquid and solid phase - Castañeda medium for Brucella spp. **colony: bacterial population derived from a single bacterial cell : pure or mixed
II. Function a. Supportive/ General purpose medium - contain nutrients that support growth of most nonfastidious organisms - without giving any particular organism a growth advantage
Culture Medium
Use Principle & Components Results
Acetate agar differential medium: E from Shigella spp
Acetate: carbon source
Amm. salt: nitrogen source
Bromthymol blue: green to blue
(+) growth: Escherichia spp. & Enterobacteriaceae
(-) growth: Shigella, Proteus, and Providencia spp. Alkaline peptone water
enrichment medium for Vibrio and Aeromonas spp. from stool specimens
alkaline pH: uninhibited replication of organisms while temporarily suppressing the replication of other organism American Trudeau Society Medium
egg-based medium used for the isolation of M. tuberculosis
eggs: provide fatty acids
potatoes: provide a carbon source.
Malachite green: inhibitory for normal bacterial flora Bacteroides Bile Esculin Agar
selective differential for isolation and identification of members of the Bacteroides fragilis group
Oxgall: separates bile-resistant species (growth) from bile-sensitive ones (no growth)
1% esculin: ability (+) or inability (-) to hydrolyze esculin
(+): dark brown or black colonies
Bile Esculin Agar
selective differential agar to isolate and identify group D streptococci and enterococci
Oxgall: inhibits the growth of most gram- positive organisms
Esculin: ability (+) or inability (-) to hydrolyze esculin
Vancomycin: detect vancomycin-resistant streptococci & enterococci
Azide: inhibit gram (-) organisms
(+): darkening of the medium
Bismuth Sulfite Agar
selective medium for the isolation of Salmonella spp.
bismuth sulfite & brilliant green: inhibits gram (+), lactose-fermenting intestinal normal microbiota & Shigella
S. typhi :
S. gallinarum, choleraesuis & Paratyphi: light green Blood Agar, Anaerobic, CDC
enriched medium useful for the isolation of fastidious anaerobes
tryptic soy agar base yeast extract, L-cysteine, hemin, sheep blood and vitamin K Blood Agar, Anaerobic, Brucella Base, Wadsworth
useful enrichment medium for the isolation of moderately fastidious, obligate anaerobes
Sheep blood: enrichment
Vitamin K1 and hemin
Blood Agar, Anaerobic, with Kanamycin and Vancomycin (KV)
selective enrichment medium rec. for the isolation of species of Bacteroides and Prevotella melaninogenica
Blood Agar, Anaerobic, Laked, with Kanamycin, Vancomycin, and Vitamin K (KVKL)
primary isolation of obligate gram-negative anaerobes, particularly Bacteroides spp.
Laked erythrocytes (lysed by freezing) & vitamin K: enrichment
Kanamycin & Vancomycin: inhibit all cocci & facultative gram (-) bacilli, except the pseudomonads
Blood Agar, Rabbit
enrichment medium in recovery and the demonstration of β- hemolysis by Haemophilus spp. and Gardnerella vaginalis Blood Agar, Sheep
routine medium to cultivate moderately fastidious organisms
infusion agar or tryptic soy agar base + 5% to 10% defibrinated sheep, rabbit, or human blood Blood Phenylethyl Alcohol Agar, Anaerobic, CDC (PEA)
selective enrichment medium for isolation of Bacteroides, Prevotella, and other obligate anaerobes from specimens containing a mixture of obligate and facultative anaerobe
yeast extract, hemin, vitamin K, and defibrinated sheep blood: enrichment
phenylethyl alcohol: inhibits facultative gram (-) anaerobes by suppressing DNA synthesis and cell division
Bordet-Gengou Blood Agar (B-G)
selective enrichment medium for the isolation of B. pertussis and B. parapertussis
Peptone: base medium
Glycerol, potato infusion, & sterile, 15% and
30% defibrinated sheep blood: enrichment
Penicillin, methicillin, or cephalexin: inhibitors Brain-Heart Infusion Broth (BHI)
enriched medium for the cultivation of non-fastidious and moderately fastidious microorganisms
rec. for the cultivation of pneumococci for the bile solubility test
Brains and beef heart: provide nutrients
Peptones, glucose, sodium chloride, and buffers
6% NaCl: diff. salt-tolerant enterococci from streptococci
Buffered Charcoal Yeast Extract Agar (BCYE)
enrichment medium useful in the isolation of Legionella spp., Francisella and Nocardia spp.
Ferric pyrophosphate: provide iron
Yeast extract, α-ketoglutarate, and L-cysteine
Activated charcoal: absorb toxic compounds Burkholderia cepacia Agar
isolate B. cepacia selectively from RT specimens from patients with cystic fibrosis
Crystal violet, bile salts, polymyxin B & ticarcillin: inhibit most gram (+) and gram (-) organisms
Inorganic salts, peptones, pyruvate, and phenol red
(+): dull yellow to hot pink medium
Campylobacter Blood Agar (Campy-BA)
selective enrichment medium for the isolation and cultivation of Campylobacter spp. from stool specimens
Brucella agar: base medium sodium bisulfite: lowers redox potential; enhancing recovery of microaerophilic organisms 10% sheep’s blood: enrichment Vancomycin: inhibit gram (+) cocci Trimethoprim: inhibit swarming strains of Proteus sp. Polymyxin B: inhibit gram (-) bacilli Amphotericin B: inhibit filamentous fungi and yeasts Cefoperazone: antipseudomonal activity and inhibit Enterobacteriaceae
Campylobacter sp.
flat, gray, nonhemolytic, raised and mucoid colonies
tan or slightly pink colonies
swarming or spreading across the surface of the plate
Campylobacter Charcoal Differential Agar
less inhibitory for Campylobacter spp. but more inhibitory for organisms found as normal fecal microbiota
Preston agar with beef extract and peptones: base medium
Cefoperazone: selection agent instead of cephazolin Campylobacter Thioglycolate Broth (Campy- Thio)
selective liquid medium used to enhance the isolation of Campylobacter spp.
holding or transport medium
Thioglycolate broth with 0% agar: base medium
Vancomycin: inhibit gram (+) cocci
Trimethoprim: inhibit swarming strains of Proteus sp.
Polymyxin B: inhibit gram (-) bacilli
Amphotericin B: inhibit filamentous fungi and yeasts
Cefoperazone: antipseudomonal activity and inhibit Enterobacteriaceae Cefsulodin- Irgasan- Novobiocin (CIN)
select for the isolation of Yersinia enterocolitica in stool samples
Peptones, beef, and yeast extracts: nutrients Sodium desoxycholate, cefsulodin, novobiocin, Irgasan, and crystal violet: inhibitor
Mannitol: differentiating agent
Neutral red: pH indicator
(+): Yersinia spp.
(+): Aeromonas spp.
Cetrimide Agar select for P. aeruginosa in specimens with mixed microbiota differentiation of non–glucose- fermenting, gram - rods
Cetrimide: inhibitory
Magnesium chloride & potassium sulfate: stimulate the production of pyocyanin
(+): P. aeruginosa Under UVL: yellow-green fluor. because of pyoverdin prod. stimulated by the low iron
promotes the development of characteristic metachromatic granules Löwenstein- Jensen Medium (LJ)
cultivate Mycobacterium spp Potato flour, egg, and glycerol: detoxify and supply nutrients required for growth
Asparagine: maximum production of niacin
Malachite green: inhibits the growth of other bacteria Lysine Iron Agar measures three parameters useful in identifying species of Enterobacteriaceae—(1) lysine decarboxylation, (2) lysine deamination, and (3) H 2 S production
Lysine: amino acid
Glucose: carbohydrate source
small amount of protein
Bromcresol purple: pH indicator
Sodium thiosulfate: sulfur source
Ferric ammonium citrate: H2S indicator MacConkey agar (MAC)
selective, differential, primary plating medium selects for Enterobacteriaceae and other gram (-) rods in the presence of mixed microbiota
bile salts & crystal violet: inhibit gram (+)
lactose: sole carbohydrate source
neutral red: pH indicator
ferric salts
(+): fermenters (pink or red colonies)
(-): non-fermenters (colorless or transparent colonies)
MacConkey Sorbitol Agar
isolate E. coli O157:H7 (doesn’t ferment sorbitol rapidly)
same components as MAC except the D- sorbitol is substituted for lactose
(+): pink to red colonies (-): colorless colonies Malonate Broth ID of species of Enterobacteriaceae, particularly Salmonella
sodium malonate: primary carbon source
small quantities of glucose and yeast extract: nutrients
bromthymol blue: pH indicator
(+): Prussian blue
(-): green
Mannitol Salt Agar
selective and differential primary culture medium useful in the recovery and ID of staphylococci from specimens with mixed microbiota
7% NaCl: inhibits most gram (-) and gram (+) bacteria except Staphylococcus spp.
Mannitol: sole carbohydrate
phenol red: pH indicator
(+): yellow (Staphylo-coccus)
(-): red
Methyl Red Voges- Proskauer Medium
Differentiating among Enterobacteriaceae
Middlebrook 7H10 and 7H Agars
cultivate Mycobacterium spp
Isoniazid-resistant strains grow better on these media, especially Middlebrook 7H
7H11: casein hydrolysate (stimulates growth of drug-resistant Mycobacterium tuberculosis)
7H10 & 7H11: amino acids, glycerol, and inorganic salts (growth factors)
OADC (oleic acid–dextrose-catalase): simulates egg components
Albumin: inhibit toxic agents
Malachite green: inhibitor Mitchison 7H Selective Agar
more selective for mycobacteria because of added antimicrobial agents
Amphotericin B, carbenicillin, polymyxin B, and trimethoprim: inhibitory to gram (-) rods in and yeast Modified Thayer-Martin Agar
selective enrichment medium used for recovering Neisseria gonorrhoeae and Neisseria meningitidis from specimens that have mixed microbiota
hemoglobin, vitamins, diphosphopyridine nucleotide, L-cysteine, NAD, and glutamine: enrichment
Cornstarch: absorb inhibitory substances that might be present
Vancomycin: inhibit the growth of gram-(+)
Colistin: inhibit gram (-) rods
Nystatin: inhibit fungi
Trimethoprim: prevents Proteus spp. from swarming Mueller-Hinton Agar
testing the susceptibility of organisms to antimicrobial agents
testing starch hydrolysis
animal infusion, casein extract, and starch: support growth of mist organism
Starch: a. protects the organisms against toxic substances b. serve as an energy source for some bacteria Mueller-Hinton Agar with 2% NaCl
detect methicillin-resistant Staphylococcus aureus (MRSA)
Mueller-Hinton Agar with 4% NaCl and 6 μg Oxacillin
screen S. aureus isolates selectively for resistance to oxacillin or nafcillin
Xylose-lysine- desoxycholate (XLD) agar
selective, differential, primary plating medium used to isolate Salmonella and Shigella spp. from stool and other specimens containing mixed biota
Sodium desoxycholate: inhibits gram (+) cocci and some gram (-) rods found in stool as normal biota
Sucrose and lactose (high conc.) Xylose (low conc.)
Phenol red: pH indicator
Lysine: detect lysine decarboxylation
Sodium thiosulfate: sulfur source
Ferric ammonium citrate:H 2 S indicator
(+)
Yellow colonies: E. coli
Yellow colonies with black centers: Citrobacter and some Proteus spp.
Colorless or red colonies: Shigella and Providencia spp.
Red colonies with black centers: Salmonella and Edwardsiella spp.
Macroscopic and Microscopic Examination
Microscopic Examination
Microscopic Description Organism Gram (+) cocci - in pairs Staphylococcus, Streptococcus, Enterococcus spp. - in terads Micrococcus, Staphylococcus, Peptostreptococcus spp. - in chain Streptococcus, Peptostreptococcus spp. - in groups Staphylococcus, Peptostreptococcus, Stomatococcus spp. - in clusters Microaerophilic Streptococcus spp., viridans streptococci, Staphylococcus spp - encapsulated Streptococcus pneumoniae, Streptococcus pyogenes (rarely), Stomatococcus mucilaginosus Gram (+) diplococci Streptococcus pneumoniae Gram (-) diplococci Neisseria spp., Moraxella catarrhalis Gram (+) bacilli - small Listeria monocytogenes, Corynebacterium spp. - medium Lactobacillus, anaerobic bacilli - large Clostridium, Bacillus spp. - diptheroid Corynebacterium, Propionibacterium, Rothia spp. - pleomorphic Gardnerella vaginalis - beaded Mycobacteria, antibiotic-affected lactobacilli, and corynebacteri - filamentous Anaerobic morphotypes, antibiotic-affected cells - filamentous, beaded branched
Actinomycetes, Nocardia, Nocardiopsis, Streptomyces, Rothia spp.
- bifid or v form Bifidobacterium spp., brevibacteria Gram (-) coccobacilli Bordetella, Haemophilus spp. (pleomorphic)
- masses Veillonella spp.
- Chains Prevotella, Veillonella spp. Gram (-) bacilli
- small Haemophilus, Legionella (thin with filaments), Actinobacillus, Bordetella, Brucella, Francisella, Pasteurella, Capnocytophaga, Prevotella, Eikenella spp.
V. Density - transparent, translucent, or opaque - requires transillumination - translucent: β-Hemolytic streptococci except group B (S. agalactiae; bull’s eye colony) - opaque: staphylococci and other gram-positive bacteria - shiny, similar to a half-pearl: Bordetella pertussis
VI. Color - white, gray, yellow, or buff - white: CoNS - gray: Enterococcus spp. - yellow or off-white: Micrococcus spp. and Neisseria - buff: Diptheroids
VII. Consistency - determined by touching the colony with a sterile loop - brittle (splinters), creamy (butyrous), dry or waxy - creamy: S. aureus - sticky: Neisseria spp. - brittle: Nocardia spp. - dry or waxy: diphtheroid - dry: β-hemolytic streptococci
VII. Pigment - P. aeruginosa: green, sometimes a metallic sheen - Serratia marcescens: brick-red - Kluyvera spp.: blue - Chromobacterium violaceum: purple - Prevotella melaninogenica: brown-black
VIII. Odor - determined when the lid of the culture plate is removed and the odor dissipates into the environment - S. aureus: old sock (stocking that has been worn continuously for a few days without washing); this odor is evident when growing on MSA - P. aeruginosa: fruity or grapelike - P. mirabilis: putrid - Haemophilus spp.: musty basement, “mousy” or “mouse nest” smell - Nocardia spp.: freshly plowed field
Growth in Liquid medium - Turbidity: refers to cloudiness of the medium resulting from growth
6. Stains and Culture-1
Course: Medical Technology (MD)
University: Our Lady of Fatima University
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