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Short answer
On a Gram stain, Haemophilus influenzae appears as small, pleomorphic gram-negative rods or coccobacilli arranged at random. The smear supports a presumptive identification only: species confirmation needs X and V factor testing on culture, and capsular type (including type b) is established by antiserum, not by the stain.
Definition and scope
The CDC Pink Book describes H. influenzae as “a fastidious, pleomorphic, gram-negative coccobacillus.” Encapsulated strains are designated serotypes a through f, and nontypeable strains, which lack a capsule, also frequently inhabit the human respiratory tract. In the laboratory the organism is often abbreviated “H. flu,” and “H flu Gram stain” and “Haemophilus influenzae type b Gram stain” are the same question at different levels of specificity: the stain itself reports shape and Gram reaction, nothing more.
This page is a laboratory and teaching reference on how the organism reads on a smear and what has to follow it. It is not clinical advice and does not cover specimen collection. For the staining method itself, see the CASRAI guide to Gram stain procedure, reagents and results.
What H. influenzae looks like on a Gram stain
The CDC/WHO manual Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae describes the organism as “small, pleomorphic Gram-negative rods or coccobacilli with random arrangements.” The Pink Book likewise says a Gram stain “may demonstrate small, gram-negative coccobacilli.” Gram-negative cells take up the counterstain rather than the crystal violet.
Counterstain choice
The same manual gives two counterstain options: safranin for 20 to 30 seconds, or carbol-fuchsin for 10 to 15 seconds. It notes that carbol-fuchsin is “considered by many to be a more effective counter-stain than safranin,” which is relevant for organisms as small and pale-staining as Haemophilus.
A sourced comparison: three organisms on a CSF Gram stain
The manual’s morphology descriptions let a reader compare what the three main meningitis bacteria look like on a stained CSF smear. Each row below is taken from that manual.
| Organism | Gram reaction and shape on the smear |
|---|---|
| Haemophilus influenzae | Gram-negative; small, pleomorphic rods or coccobacilli, random arrangement |
| Neisseria meningitidis | Gram-negative; coffee-bean-shaped diplococci, intracellular and extracellular in polymorphonuclear cells |
| Streptococcus pneumoniae | Gram-positive; lanceolate diplococci, sometimes in short chains |
Two of the three are gram-negative, so the Gram reaction alone cannot separate H. influenzae from N. meningitidis; shape and arrangement do the work, and culture settles it.
What the Gram stain does not tell you
The manual states that a presumptive diagnosis of bacterial meningitis caused by H. influenzae, S. pneumoniae or N. meningitidis “can be made by Gram stain of the CSF sediment or by detection of specific antigens,” and that a positive result can be evidence of infection “even if cultures fail to grow.” Presumptive is the operative word. Identification of the isolate follows a separate path:
- Growth requirements. The Pink Book states that the organism “requires hemin (X factor) and nicotinamide-adenine-dinucleotide (NAD, also known as V factor) for in vitro growth.” The manual says that determination of X and V factor requirements “is necessary to confirm the identity of the isolate,” and that H. influenzae grows only around the disk containing both factors.
- Plating. Specimens are inoculated onto both a supplemented chocolate agar plate and a blood agar plate. A small gram-negative coccobacillus that needs X and V factors, grows on chocolate agar and not on blood agar, and has a pungent indole smell may be H. influenzae. Growth on chocolate agar is explained by haemin released during the heating used to prepare it.
- Other species. H. haemolyticus is described as an oral species that also requires X and V factors, so those factors alone do not separate it; the manual differentiates it by growth on heart infusion or blood agar with 5% horse blood.
- Culture. The Pink Book states that “a positive culture for H. influenzae establishes the diagnosis,” and that all isolates should be serotyped.
Haemophilus influenzae type b versus nontypeable strains on a Gram stain
A type b organism and a nontypeable organism look the same on a Gram stain. Capsule type is not visible on the smear. In the manual’s serotyping scheme a suspected type b isolate is tested with type b antiserum; if that is non-reactive, a polyvalent antiserum is used, and an isolate that is negative with both is “probably non-typable.”
The manual adds that “in the absence of vaccination, almost all cases of Haemophilus influenzae meningitis are caused by serotype b.” The Pink Book notes that nontypeable strains are generally less virulent than encapsulated strains among previously healthy individuals, and that otitis media and bronchitis are generally caused by nontypeable strains. Which type a laboratory is looking at therefore matters for interpretation, and the stain cannot supply it.
Where it matters for laboratories
For a teaching or clinical microbiology laboratory, the practical points from the manual are narrow and sourced: a reader should expect small pleomorphic gram-negative forms, should not treat the smear as species identification, and should keep the culture, X and V factor and serotyping steps in the written procedure. The manual also specifies that CSF is held at room temperature before Gram staining. Laboratories that document these steps in controlled procedures can use the CASRAI entry on standard operating procedures as a reference point, and the entry on biosafety levels for the containment framework used in microbiology laboratories. For broader background, see what bacteriology is.
Common misunderstandings
- “Gram-negative means it is H. influenzae.” No. N. meningitidis is also gram-negative; the two are separated by morphology on the smear and by culture.
- “The name means it causes influenza.” The Pink Book describes a bacterium, not the influenza virus.
- “The stain tells you it is type b.” It does not. Typing requires antiserum.
- “No growth in culture cancels a positive smear.” The manual notes a positive Gram stain or antigen result can provide evidence of infection even when cultures fail to grow.
FAQ
Is Haemophilus influenzae gram-positive or gram-negative?
Gram-negative. The CDC describes it as a fastidious, pleomorphic gram-negative coccobacillus.
What does an H flu Gram stain look like?
Small, pleomorphic gram-negative rods or coccobacilli in random arrangement, as described in the CDC/WHO meningitis laboratory manual.
Why is chocolate agar used for H. influenzae?
The organism needs X factor (hemin) and V factor (NAD) to grow. The manual explains that chocolate agar supplies haemin released during heating, so the organism grows on chocolate agar but not on blood agar.
Can a Gram stain identify Haemophilus influenzae type b?
No. Type b and nontypeable strains look alike; type is determined by serotyping with antiserum.
Is a Gram stain enough to confirm the species?
No. It supports a presumptive identification; the manual requires X and V factor testing to confirm the isolate.
Sources
- CDC, Epidemiology and Prevention of Vaccine-Preventable Diseases (Pink Book), Chapter 8: Haemophilus influenzae. https://www.cdc.gov/pinkbook/hcp/table-of-contents/chapter-8-haemophilus-influenzae.html. Date checked: 2026-10-29.
- WHO/CDC, Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae (WHO/CDS/CSR/EDC/99.7, 1999). https://www.who.int/docs/default-source/documents/publications/laboratory-methods-for-diagnosis-of-meningitis.pdf. Date checked: 2026-10-29.
