MODIFIED ALP and NON-FERMENTER IDENTIFICATION MEDIA

Cat. no. Y21 FID (Fluorescence-Indole-Denitrification), 13x100mm Tube,
4.5ml Slant
10 tubes/box
Cat. no. Y22 FLN (Fluorescence-Lactose-Denitrification), 13x100mm Tube,
3.8ml Slant
10 tubes/box
Cat. no. Y23 Motility Nitrate, 13x100mm Tube, 3ml Deep 10 tubes/box
Cat. no. Y61 Modified ALP Acetamide, 13x100mm Tube, 3ml Slant 10 tubes/box
Cat. no. Y62 Modified ALP Arabinose, 13x100mm Tube, 3ml Slant 10 tubes/box
Cat. no. Y63 Modified ALP Dextrose, 13x100mm Tube, 3ml Slant 10 tubes/box
Cat. no. Y66 Modified ALP Maltose, 13x100mm Tube, 3ml Slant 10 tubes/box
Cat. no. Y67 Modified ALP Mannitol, 13x100mm Tube, 3ml Slant 10 tubes/box
Cat. no. Y68 Modified ALP Lactose, 13x100mm Tube, 3ml Slant 10 tubes/box
Cat. no. Y69 Modified ALP Sucrose, 13x100mm Tube, 3ml Slant 10 tubes/box
Cat. no. Y70 Modified ALP Urea, 13x100mm Tube, 3ml Slant 10 tubes/box
Cat. no. Y71 Modified ALP Xylose, 13x100mm Tube, 3ml Slant 10 tubes/box

INTENDED USE

Hardy Diagnostics Modified ALP (Aerobic Low Peptone) Media, also known as OLP (Oxidative Low Peptone), are used to aid in the identification of non-fermentative gram-negative bacilli (NFB). When Modified ALP substrates are included with a battery of other properly selected tests or substrates, the most commonly encountered NFB can be identified within 24 hours.

Fluorescence-Denitrification (FN) Media are used to aid in the detection of fluorescein pigment and complete reduction of nitrate to nitrogen gas. The medium, if supplemented with tryptophane (FID), aides in the detection of indole-producing microorganisms. The addition of lactose and a pH indicator (FLN) allow for the detection of lactose-utilizing microorganisms.

Motility Nitrate is used to detect nitrate reduction and motility of NFB.

SUMMARY

Aerobic Low Peptone (ALP) Media, a modification of Oxidative-Fermentative (OF) Media and Buffered Single Substrate (BSS) Media, were originally formulated by Pickett and Greenwood to demonstrate the rapid acidification or alkalization of substrates by non-fermentative, gram-negative bacilli (NFB). (3,5)

As compared to OF Media, Pickett and Greenwood's media contains a reduced (25% less) amount of peptone. The limited amount of peptone minimizes the chance for alkalization to occur which can neutralize weak acid producers.

Hardy Diagnostics Modified ALP Media is a modification of the formula developed by Greenwood and Pickett. Like ALP Media, the modified formula employs phenol red as the color indicator, and depending upon the pH adjustment of the basal medium, Modified ALP Media can be used to detect acidification of carbohydrates or alkalization of organic salts and amides.

Fluorescence-Denitrification (FN) Media are formulated to detect fluorescein pigment and complete reduction of nitrate to nitrogen gas. These two characteristics are important in the identification of the pseudomonads and other non-fermentative bacilli.

Fluorescence-Indole-Denitrification (FID) and Fluorescence-Lactose-Denitrification (FLN) Media are modifications of FN Media. FLN incorporates lactose and phenol red indicator in the formula to allow detection of acid and lactose utilization. FLN is useful in the identification of the non-fermenters that are strongly lactose-positive. FID incorporates the use of tryptophane to allow the detection of organisms which possess the enzyme tryptophanase. Tryptophanase degrades tryptophane which results in the production of indole, pyruvic acid, and ammonia.

Motility Nitrate Media is used to detect motility and nitrate reduction of NFB. Motility is observed macroscopically by the appearance of a diffuse zone of growth spreading from a line of inoculation. Non-motile organisms grow only along the stab line and leave the surrounding medium clear. Nitrate reduction is manifested by cracks in the agar produced by the presence of gas bubbles.

FORMULA

Ingredients per liter of deionized water:*

Modified ALP
Modified ALP Media is a proprietary medium which includes a buffered medium of inorganic salts in a nutrient base with agar and phenol red.
The ALP Media used to determine alkalization of organic salts and amides (such as urea and acetamide) consists of the basal medium, 1% salt or amide, and 0.1% dextrose. The final pH is adjusted to 6.5 +/- 0.2 at 25ºC.
The ALP Media used to determine acidification of carbohydrates consists of the basal medium and 1% carbohydrate. The final pH is adjusted to 7.6 +/- 0.2 at 25ºC.
FID (Fluorescence-Indole-Denitrification) FLN (Fluorescence-Lactose-Denitrification)
Tryptone 10.0gm Lactose 20.0gm
Proteose Peptone 10.0gm Proteose Peptone 10.0gm
Potassium Nitrate 2.0gm Potassium Nitrate 2.0gm
Potassium Phosphate 1.5gm Magnesium Sulfate 1.5gm
Magnesium Sulfate 1.5gm Potassium Phosphate 1.5gm
Tryptophane 1.0gm Potassium Nitrite 0.5gm
Glycerol 10.0ml Dextrose 0.5gm
Agar 15.0gm Sodium Acetate Trihydrate 0.5gm
Phenol Red 18.0mg
Agar 15.0gm
Final pH of 7.2 +/- 0.2 at 25ºC. Final pH of 7.2 +/- 0.2 at 25ºC.
Motility Nitrate
Pancreatic Digest of Casein 10.0gm
Peptic Digest of Animal Tissue 5.0gm
Sodium Chloride 5.0gm
Yeast Extract 3.0gm
Potassium Nitrate 3.0gm
Beef Heart Infusion 2.0gm
Agar 3.0gm
Final pH of 7.4 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Upon receipt store media at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed.

PRECAUTIONS

PROCEDURE

Specimen Collection: These media are not intended for use as primary isolation media. They are for use in characterizing pure cultures. Consult listed references for information regarding the processing and inoculation of specimens. (2-5)

Refer to the keywords " Specimen Collection and Transport " and " Specimen Collection Chart " on the Hardy Diagnostics Technical Document website for more information on specimen collection methods.

Method of Use:

1. Inoculate a KIA (Cat. no. L70 ) or TSI (Cat. no. L50 ) slant to determine if an isolate is an NFB.

Note: Refer to the Instructions for Use (IFU) documents for KIA or TSI for procedural use and interpretation of results for each medium.

2. Following 24 hours of incubation of KIA or TSI, non-fermentative bacilli (NFB) present as either 'alkaline' over 'no change'; or 'no change' over 'no change'. Slow growing NFB may require an additional 24 hour incubation period before reactions can be interpreted. When KIA or TSI are used to determine whether or not the isolate is an NFB, there is no need to use the open or closed OF tubed method to determine the organism's ability to ferment or oxidize.

3. Perform an oxidase test (Cat. no. Z93 ) using an inoculum from the 24 hour growth on the KIA or TSI used in step 1 above.

4. Depending upon the results of the oxidase reaction, inoculate the media as listed below according to the following methods of inoculation:

METHOD OF INOCULATION

The following media should be inoculated for identification of:

OXIDASE-NEGATIVE NFB OXIDASE-POSITIVE NFB
Cat. no. Y22 FLN Cat. no. Y21 FID
Cat. no. Y23 Motility Nitrate Cat. no. Y22 FLN
Cat. no. Y62 Modified ALP Arabinose Cat. no. Y23 Motility Nitrate
Cat. no. Y69 Modified ALP Sucrose Cat. no. Y61 Modified ALP Acetamide
Cat. no. Y70 Modified ALP Urea Cat. no. Y63 Modified ALP Dextrose

Modified ALP Media:

1. Using a heavy inoculum of test isolate, spot inoculate (do not streak) one area on the surface of the slant.

Note: A spot application of the inoculum, rather than a streak across the entire surface, introduces a high concentration of preformed enzymes or other metabolic products than can be detected more quickly by the endpoint indicator. (3)

2. Incubate at 35ºC. for 24-48 hours*. Modified ALP Media with organic salts or amides, such as acetamide, should be incubated 72 hours before negative alkalization results are interpreted.

Note: Some organisms such as Burholderia cepacia grow better (produce stronger reactions) when grown at room temperature on modified ALP with carbohydrates.

Motility Nitrate Media:

1. Using growth from an 18-24 hour pure culture, inoculate the medium by stabbing the agar to a depth of 2-3mm.

2. Incubate at 35ºC. for 24-48 hours.

FLN (Fluorescence-Lactose-Denitrification) Media:

1. Using growth from an 18-24 hour pure culture, stab to the bottom of the tube and streak the agar surface as the needle is withdrawn from the butt of the medium.

2. Incubate at 35ºC. for 24-48 hours.

FID (Fluorescence-Indole-Denitrification) Media:

1. Using growth from an 18-24 hour pure culture, stab to the bottom of the tube and streak the agar surface as the needle is withdrawn from the butt of the medium.

2. Incubate at room temperature (15-30ºC.) for 24-48 hours.

3. Add 4 drops of Indole Kovacs Reagent (Cat. no. Z67) to determine Indole reaction.

INTERPRETATION OF RESULTS

Test Media Negative Reaction Positive Reaction
Modified ALP Salts
(e.g. Acetamide and Urea) Yellow to yellow orange slant Red slant
Modified ALP Sugars
(e.g. Arabinose, Dextrose, Lactose, Maltose, Mannitol, Sucrose, and Xylose) Red slant Yellow slant
Motility Nitrate
Motility No motility, growth confined "Puff ball" of motility at 4 hours or turbid tube
Denitrification Agar intact, no bubbles Agar broken with gas bubbles
FID
Fluorescence No fluorescence with UV light Fluorescence with UV light
Indole No color change when 4 drops of indole reagent are added Red reaction when 4 drops of indole reagent are added
Denitrification Agar intact, no bubbles Agar broken with gas bubbles
FLN
Fluorescence
No fluorescence with UV light Fluorescence with UV light
Lactose-Acidification Red slant Yellow slant
Denitrification Agar intact, no bubbles Agar broken with gas bubbles

LIMITATIONS

Some NFB are slow acid producers. Modified ALP Media with carbohydrates should be incubated 48 hours before negative acidification results are interpreted.

Modified ALP Media with organic salts or amides, such as acetamide, should be incubated 72 hours before negative alkalization results are interpreted.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, incinerator, incubators, pasteur pipets, as well as serological and biochemical reagents, are not provided. Additionally, the identification method described in this technical information sheet requires OxiStrips (Cat. no. Z93 ), Kovacs Indole Reagent ( Cat. no. Z67 ), and a long-wave UV lamp (Cat. no. UVL56).

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Modified ALP with Acetamide
Pseudomonas aeruginosa
ATCC ® 27853
E 24-72hr 35°C Aerobic Growth; color change from yellow to red
Moraxella ( Branhamella ) catarrhalis
ATCC ® 25240
E 24-72hr 35°C Aerobic Growth; no color change
Modified ALP with Urea
Pseudomonas aeruginosa
ATCC ® 27853
E 24-48hr 35°C Aerobic Growth; color change from yellow to red
Burkholderia ( Pseudomonas ) cepacia
ATCC ® 25416
E 24-48hr 35°C Aerobic Growth; no color change
Modified ALP with Maltose
Burkholderia ( Pseudomonas ) cepacia
ATCC ® 25416
E 24-48hr 35°C** Aerobic Growth; color change from red to yellow
Moraxella ( Branhamella ) catarrhalis
ATCC ® 25240
E 24-48hr 35°C Aerobic Growth; no color change
Modified ALP with Arabinose, Dextrose, Mannitol, and Xylose
Pseudomonas aeruginosa
ATCC ® 27853
E 24-48hr 35°C Aerobic Growth; color change from red to yellow
Moraxella ( Branhamella ) catarrhalis
ATCC ® 25240
E 24-48hr 35°C Aerobic Growth; no color change
Modified ALP with Lactose, and Sucrose
Burkholderia ( Pseudomonas ) cepacia
ATCC ® 25416
E 24-48hr 35°C** Aerobic Growth; color change from red to yellow
Pseudomonas aeruginosa
ATCC ® 27853
E 24-48hr 35°C Aerobic Growth; no color change
Motility Nitrate
Pseudomonas aeruginosa
ATCC ® 27853
D 24-48hr 35°C Aerobic Growth; positive motility and denitrification
Moraxella ( Branhamella ) catarrhalis
ATCC ® 25240
D 24-48hr 35°C Aerobic Growth; negative motility and denitrification
FLN (Fluorescence-Lactose-Denitrification)
Pseudomonas aeruginosa
ATCC ® 27853
C 24-48hr 35°C Aerobic Growth; positive fluorescence (blue) and denitrification (gas bubbles formed)
Burkholderia ( Pseudomonas ) cepacia
ATCC ® 25416
C 24-48hr 35°C Aerobic Growth; negative fluorescence and denitrification, lactose-positive (turns from red to yellow)
FID (Fluorescence-Indole-Denitrification)
Pseudomonas aeruginosa
ATCC ® 27853
C 24-48hr 15-30°C Aerobic Growth; positive fluorescence (blue) and denitrification (gas bubbles formed), negative for indole
Burkholderia ( Pseudomonas ) cepacia
ATCC ® 25416
C 24-48hr 15-30°C Aerobic Growth; negative fluorescence, denitrification, and indole
Chryseobacterium ( Flavobacterium ) meningosepticum
ATCC ® 13253
C 24-48hr 15-30°C Aerobic Growth; indole-positive (turns red when 4 drops of indole reagent are added), fluorescence and denitrification-negative

** Some organisms such as Burholderia cepacia grow better (produce stronger reactions) when grown at room temperature on modified ALP with carbohydrates.

User Quality Control

PHYSICAL APPEARANCE

P. aeruginosa growing on Modified ALP with Acetamide

Pseudomonas aeruginosa (ATCC ® 27853) growing on Modified ALP with Acetamide (Cat. no. Y61). Incubated aerobically for 24 hours at 35ºC. The red color deveopment was indicative of a positive acetamide reaction.

B. catarrhalis growing on Modified ALP with Acetamide

Moraxella (Branhamella) catarrhalis (ATCC ® 25240) growing on Modified ALP with Acetamide (Cat. no. Y61). Incubated aerobically for 24 hours at 35ºC. No red color development was indicative of a negative acetamide reaction.

Modified ALP wth Acetamide

Uninoculated tube of Modified ALP with Acetamide (Cat. no. Y61)






P. aeruginosa growing on Modified ALP with Urea

Pseudomonas aeruginosa (ATCC ® 27853) growing on Modified ALP with Urea (Cat. no. Y70). Incubated aerobically for 24 hours at 35ºC. The pink-red color development was indicative of a positive reaction.

B. cepacia growing on Modified ALP with Urea

Burkholderia (Pseudomonas) cepacia (ATCC ® 25416) growing on Modified ALP with Urea (Cat. no. Y70). Incubated aerobically for 24 hours at 35ºC. No pink-red color development was indicative of a negative reaction.



Modified ALP with Urea

Uninoculated tube of Modified ALP with Urea (Cat. no. Y70).








B. cepacia growing on Modified ALP with Maltose

Burkholderia (Pseudomonas) cepacia (ATCC ® 25416) growing on Modified ALP with Maltose (Cat. no. Y66). Incubated aerobically for 24 hours at 35ºC. The yellow color development was indicative of a positive maltose reaction.

B. catharrhalis growing on Modified ALP with Maltose

Moraxella (Branhamella) catarrhalis (ATCC ® 25240) growing on Modified ALP with Maltose (Cat. no. Y66). Incubated aerobically for 24 hours at 35ºC. No yellow color development was indicative of a negative maltose reaction.



Modified ALP with Maltose

Uninoculated tube of Modified ALP with Maltose (Cat. no. Y66).



BIOCHEMICAL PROFILES OF COMMONLY ENCOUNTERED NFB

The ALP Identification System is designed to identify the following non-fermenters:

Oxidase-Positive:


P.
aeruginosa
Chryseo-
bacterium

spp.
P.
putida
P.
stutzeri
Ralstonia
pickettii
Delftia
acidovorans
B.
bronchiseptica
S.
paucimobilis
B.
cepacia

complex
Motility Nitrate (Y23)
Motility + - + + + + + - +
Nitrate + - - (-) (-) - - - -
FID (Y21) and FLN (Y22)
Fluorescence + - + or - - - - - - -
Indole - + - - - orange
indole
- - -
Lactose - - - - - - - + +
Denitrification + - - - - NA NA NA NA
Modified ALP Dextrose (Y63)


+ + or - + + + - - + +
Modified ALP Acetamide (Y61)


+ - - - - + - - (+)

Oxidase-Negative:


A.
anitratus
S.
maltophilia
A.
lwoffi
C.
luteola
F.
oryzihabitans
Motility Nitrate (Y23)
Motility - + - + +
Nitrate - - - - -
FLN (Y22)
Fluorescence - - - - -
Indole - - - - -
Lactose + - - - -
Denitrification - - - NA NA
Modified ALP
Sucrose (Y69) - + or - - (-) (-)
Arabinose (Y62) + - - + +
Urea (Y70) + or - - - (-) (-)

NA = Results not available - = 10% or less of the strains are positive
+ = 90% or more of the strains are positive (-) = 11-50% of the strains are positive
(+) = 51-89% of the strains are positive

FLN is incubated at 35ºC.
FID is incubated at room temperature (15-30ºC.).

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

3. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

4. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

5. Gilardi, G.L., ed. 1985. Non-Fermentative Gram-Negative Rods, Vol. 16. Marcel Dekker, Inc., New York, NY.


ATCC is a registered trademark of the American Type Culture Collection.

032816gr