Cat. no. Z88 Methylene Blue, Loefflers 15ml


Hardy Diagnostics Methylene Blue, Loefflers stain is a multipurpose stain recommended for use in staining bacteria and leukocytes.


Methylene Blue, Loefflers is recognized as a simple stain used for determining bacterial morphology. It is also useful as an aid in interpretation of the Quellung test.(6) It is a cationic dye which stains the cell a blue color. The presence of negatively charged molecules in the cell causes the staining phenomenon, as the positively charged dye is attracted to negatively charged particles, such as polyphosphates like DNA and RNA.(6)

Methylene Blue, Loefflers may be used in the presumptive identification of Corynebacterium diphtheria. Metachromasia, a condition characteristically seen in C. diphtheria, is due to an accumulation of polymerized polyphosphates in high concentration inside the cell. This condition appears as polyphosphate granules stained deeply blue, surrounded by lighter blue stained cytoplasm, and are often called Babes-Ernst bodies or metachromatic granules.(5,6) The cell itself is characterized by a banded or beaded appearance.(5,6)

Methylene Blue, Loefflers is also recommended for use in the staining of gram-negative bacteria found in spinal fluid, namely Haemophilus influenzae and Neisseria meningitidis.(5) This technique, more so than the gram stain, allows for better contrast between these gram-negative organisms and the background.

In addition to the staining of microorganisms, Methylene Blue, Loefflers was demonstrated by Harris in 1972 to be an effective stain for the enumeration of leukocytes in mucus or feces.(3)


Ingredients per liter of deionized water:*

Methylene Blue, Certified 3.0gm
Potassium Hydroxide, 10% 1.0ml
Ethanol, 95% 300.0ml

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


Upon receipt store at 2-30ºC. Product should not be used if there are any signs of deterioration or if the expiration date has passed. Do not expose to excessive heat or moisture.


Warning! Causes Irritation. Avoid contact with eyes, skin and mucous membranes. If contact with eyes occurs, flush immediately with large amounts of water for at least 15 minutes, and call a physician. Flush skin with water. Wash clothing before reuse.

Warning! Staining solutions are hazardous in nature. Wear appropriate safety apparel when working with staining solutions. It is recommended that staining procedures be performed under a hood and with adequate ventilation.


Specimen Collection: Consult listed references for information on specimen collection.(1-4)

Simple Stain:
Prepare and heat or methanol fix a smear of the organism to be examined on a clear glass slide. Flood the slide with the Methylene Blue, Loefflers stain. Allow to stand for one to three minutes. Rinse slide with tap water, and blot dry. Examine the smear at 1000X, using the oil immersion objective.

Staining of C. diphtheriae:
Prepare and heat or methanol fix a smear of C. diphtheriae grown on Loefflers medium. Stain the slide as described above.

Staining of Leukocytes:
Place a drop of liquid feces or a small fleck of mucus on a clean glass slide with a wooden applicator stick. Add two drops of Methylene Blue, Loefflers. Mix thoroughly and carefully, and place a coverslip on the mixture. To obtain good nuclear staining, let the slide stand for two to three minutes. Under low power (100X), make rough quantitative counts by approximating the average number of leukocytes and erythrocytes per field. Differential counts should be performed under high power (400X), counting 200 cells when possible. Only cells clearly identifiable as mononuclear or polymorphonuclear are to be included in the differential count. Epithelial cells and macrophages that cannot be clearly identified are to be ignored. (3)


When observed microscopically using oil immersion, bacterial cells stain a medium blue color, and the background stains lighter blue. When C. diphtheriae cells are stained, they appear banded or beaded with deep blue staining metachromatic granules. Leukocytes appear light blue (cell cytoplasm) with a dark blue nuclei. Consult appropriate references for more details. (3,7)


Morphology of C. diphtheriae is most distinctive when grown on Loefflers medium.(2) If C. diphtheriae is grown on other media, less satisfactory results may be seen.

When staining C. diphtheriae, overstaining may reduce the contrast between the bacteria and background, or between the cytoplasm and granules.(5)

Some strains of Propionibacterium, Actinomyces, and pleomorphic forms of streptococci may mimic the characteristic stained appearance of C. diphtheriae.(5)


Standard microbiological supplies and equipment such as loops, glass slides, coverslips, immersion oil, culture media, pipettes, applicator sticks, microscopes, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Results
Escherichia coli
ATCC® 25922
Stains blue in color
Staphylococcus aureus
ATCC® 25923
Stains blue in color
Corynebacterium diphtheriae
ATCC® 8028
Cell appears banded or beaded with deep blue granules and a lighter blue cytoplasm

User Quality Control

It is recommended that each new lot of stain be tested with known positive and negative controls and retested each day of use thereafter.(1,4,8)

The microscope should be calibrated (within the last 12 months), and the objectives and oculars used for the calibration procedure should be in place on the microscope when objects are measured.(1,4)

It is recommended that positive controls be run in parallel with patient specimens and that results from this staining procedure be reported only if positive control smears are acceptable.

Physical Appearance

Methylene Blue, Loefflers stain should appear dark royal blue in color.


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. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Henry, J.B., ed. 1979. Clinical Diagnosis and Management, 16th ed., Vol. I. W.B. Saunders Company, Philadelphia, PA.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

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

6. Lillie, R.D. 1977. H.J. Conn's Biological Stains, 9th ed. Williams & Wilkins Company, Baltimore, MD. Reprint by Sigma Chemical Company, 1991.

7. Tilton, R.C., A. Balows, et al. 1992. Clinical Laboratory Medicine. Mosby Year Book, St. Louis, Missouri.

8. State Operation Manual, Provide Certification. 1991. Department of Health and Human Services, Berkeley, CA.

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