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CNA agar is a Columbia-base blood agar made selective for Gram-positive bacteria by adding two antibiotics, colistin and nalidixic acid (the “CNA”). It is also called Columbia CNA agar, agar CNA or CNA blood agar. Because makers set the antibiotic and agar levels differently, a plate labelled “CNA” from one supplier is not automatically the same specification as another supplier’s.
Short answer
CNA agar is a selective and differential plate: colistin and nalidixic acid suppress many Gram-negative bacteria, while sheep blood supports growth of fastidious Gram-positive organisms and shows haemolytic reactions. Per the Bio-Rad instructions for use, it is intended for mixed-flora specimens such as genital tract or oropharyngeal samples. Per the BD instructions for use, it is for isolating and differentiating Gram-positive organisms from clinical and nonclinical material.
Definition and scope
CNA agar belongs to the family of blood agar plates described in what is an agar plate, where it sits in the selective-and-differential group. The name comes from the selective mixture, not from a different nutrient base. Bio-Rad describes the product as “Columbia CNA + 5% sheep blood” and gives the intended use as a selective medium for Gram-positive bacteria that is also used to demonstrate haemolytic reactions. BD describes its BBL Columbia CNA Agar with 5% Sheep Blood as a selective and differential medium for the isolation and differentiation of Gram-positive microorganisms.
The formula is credited in the Bio-Rad insert to Ellner and colleagues (Ellner PD, Stoessel CJ, Drakeford E, Vasi F. “A new culture medium for medical bacteriology.” Am J Clin Pathol 1966;45:502-504). According to the BD insert, Ellner found that colistin at 10 mg per litre and nalidixic acid at 15 mg per litre in a Columbia base with 5% sheep blood supported staphylococci, haemolytic streptococci and enterococci while inhibiting Proteus, Klebsiella and Pseudomonas.
How CNA agar works in practice
The BD insert states that colistin disrupts the cell membranes of Gram-negative organisms, whereas nalidixic acid blocks DNA replication in susceptible Gram-negative bacteria. The Columbia base and sheep blood then favour growth of most Gram-positive organisms and let the reader see haemolysis. The same insert notes that sheep blood supplies X factor (haem) but not V factor (NAD), because of NADase activity.
Both manufacturers’ instructions give the expected appearance of typical Gram-positive growth. BD lists non-group D streptococci as small white-gray colonies with beta or alpha haemolysis, enterococci as small blue-gray, staphylococci as large white-gray or cream-yellow with variable haemolysis, and Candida as small white colonies. Bio-Rad describes the uninoculated plate as a cherry-red agar.
How two makers’ CNA plates differ
The figures below are those stated in each maker’s instructions for use. They are the main reason to treat “CNA” as a family of related formulations rather than one fixed specification.
| Item | Ellner original (as reported in BD insert) | Bio-Rad Columbia CNA + 5% sheep blood | BD BBL Columbia CNA with 5% sheep blood |
|---|---|---|---|
| Colistin | 10 mg/L | 0.010 g/L | 10 mg/L |
| Nalidixic acid | 15 mg/L | 0.015 g/L | 10 mg/L (reduced to improve recovery of Gram-positive cocci from clinical specimens) |
| Blood | 5% sheep | 5% defibrinated sheep | 5% sheep |
| Incubation stated in insert | Not stated | 24-48 h at 37 °C, normal atmosphere or CO2 | 24-48 h at 35 ± 2 °C with 3.5% CO2 |
| Pack in the insert | Not applicable | Box of 20 plates, 90 mm (code 63954) | 20 plates (cat. 251352) |
What grows and what is held back
Each insert lists quality control organisms. The panels differ by maker, and the same species can carry a different ATCC strain number, so each is shown under its own maker.
| Maker | Expected growth | Expected no or partial growth |
|---|---|---|
| Bio-Rad | Streptococcus pyogenes ATCC 19615 (good growth, haemolysis); S. pneumoniae ATCC 6303 (good growth, haemolysis); Enterococcus faecalis var. zymogenes ATCC 29212 (good growth, no haemolysis) | Neisseria meningitidis ATCC 13090 (with CO2), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853: no growth |
| BD (CLSI strains) | S. pyogenes ATCC 19615 (growth, beta haemolysis); S. pneumoniae ATCC 6305 (growth, alpha haemolysis); Staphylococcus aureus ATCC 25923 (growth) | Proteus mirabilis ATCC 12453: partial inhibition |
Where it matters for labs and clinics
The practical consequences come straight from the manufacturers’ stated limits.
- Selectivity is not absolute. Bio-Rad states that some Enterobacteriaceae, such as Proteus, Serratia, Pseudomonas and Klebsiella, are resistant to the CNA antibiotic mixture and may not be inhibited. An excessively heavy inoculum also decreases the inhibitory effect.
- Some Gram-positive strains may grow poorly. Bio-Rad notes that some strains may not grow because of their nutritional needs, and that colonies are generally smaller than on Columbia sheep blood agar without CNA.
- One plate is not enough. BD states that a single medium is rarely adequate for detecting all organisms of potential significance and that results should be compared with non-selective cultures. Identification of species needs complementary tests, and organisms must be in pure culture for identification.
- Haemolysis can mislead. BD warns that the relatively high carbohydrate content can make beta-haemolytic streptococci produce a greenish reaction that may be mistaken for alpha haemolysis. See alpha haemolysis on a blood agar plate for how that reaction is described.
Checklist for specifying and receiving CNA plates
Each point below is taken from the Bio-Rad or BD insert; neither manufacturer states these as a purchasing rule, so treat them as a way of reading the label against the insert.
- Confirm the maker’s name and product code, and read that maker’s insert, since formulations differ (nalidixic acid 15 mg/L versus 10 mg/L in the table above).
- Note the stated storage temperature: both makers give 2-8 °C for the plates. BD adds storage in the dark, no freezing or overheating, and warming to room temperature before use.
- Check the maker’s quality control strains and match them to your own QC records (the ATCC numbers differ between the two inserts).
- BD advises discarding plates that show contamination, discoloration, drying or cracking.
For other plate formats and what the Petri dish itself is, see what is a Petri dish; general bench handling is in the aseptic technique guide.
Common misunderstandings
- “CNA agar kills all Gram-negative bacteria.” The Bio-Rad insert names Proteus, Serratia, Pseudomonas and Klebsiella as genera that may not be inhibited, and BD reports partial rather than complete inhibition for Proteus mirabilis in its QC panel.
- “CNA blood agar is the same as plain blood agar with antibiotics added by the user.” Both inserts describe a manufactured, quality-controlled product with a stated formula; the formulas differ between makers.
- “Haemolysis on CNA reads the same as on plain blood agar.” BD cautions about greenish reactions from beta-haemolytic streptococci, and Bio-Rad notes smaller colonies.
- “A growth result identifies the organism.” Both makers state that further tests are required for identification.
FAQ
What does CNA stand for in CNA agar?
CNA refers to the colistin and nalidixic acid selective mixture added to a Columbia base with sheep blood. The BD and Bio-Rad inserts both describe this mixture as the source of selectivity.
Is agar CNA the same as CNA blood agar?
The names describe the same plate type: Columbia agar base with 5% sheep blood and colistin plus nalidixic acid. The manufacturers’ product names are “Columbia CNA + 5% sheep blood” (Bio-Rad) and “Columbia CNA Agar with 5% Sheep Blood” (BD BBL).
What is CNA blood agar used for?
Per the Bio-Rad insert, it is a selective medium for Gram-positive bacteria that also demonstrates haemolytic reactions, intended for mixed-flora specimens such as genital tract or oropharyngeal samples. BD describes it as a selective and differential medium for isolating and differentiating Gram-positive organisms from clinical and nonclinical materials.
Sources
- Bio-Rad, instructions for use 63954, “Columbia CNA + 5% sheep blood” (2015/05): commerce.bio-rad.com insert 63954. Checked 29 October 2026.
- BD BBL Columbia CNA Agar with 5% Sheep Blood, insert 111-251352-N-00 (September 2014): bdj.co.jp insert 111-251352-N-00. Checked 29 October 2026.
- Ellner PD, Stoessel CJ, Drakeford E, Vasi F. A new culture medium for medical bacteriology. Am J Clin Pathol. 1966;45:502-504, as cited in the Bio-Rad and BD inserts.
