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General laboratory information for lab and clinic staff, drawn from the sources listed below. It is not medical advice and not a bench procedure.
Short answer
Motility test medium is a semisolid agar, made with a low agar concentration, that is used to determine whether a microorganism is motile (ASM, 2016). Growth that spreads diffusely outward from the line of inoculation, or turbidity through the medium, indicates motility; growth only along the inoculation line indicates a non-motile result (CDC REACH). “Motility agar” is the same idea under another name.
Definition and scope
The American Society for Microbiology (ASM) protocol states that “motility test medium is used to determine the motility of microorganisms.” The CDC REACH core microbiology job aid describes the medium the same way: “Motility media is a semi-solid media which is the result of a low concentration of agar.” The softness is the point. A firm agar holds cells where they were placed, while a soft agar lets motile cells move through it.
Motility testing is also built into larger multitest media. ASM lists motility-indole-lysine, motility-indole-ornithine and sulfide-indole-motility as examples used for Enterobacteriaceae. This page covers the stand-alone semisolid motility medium and how it relates to those combined media; it does not give a step-by-step inoculation procedure.
How semisolid motility medium works in practice
Why a soft agar shows motility
ASM notes that the most common mechanism of bacterial motility involves flagella. In a semisolid column, flagellated cells can swim away from where they were introduced, so the growth zone widens beyond the original line. ASM traces the macroscopic approach to Tittsler and Sandholzer, whose semisolid agar showed “over 99% concordance” with the hanging-drop method, with motility read as diffuse spread of growth beyond the stab line.
Why tetrazolium (TTC) is sometimes added
Kelly and Fulton added triphenyltetrazolium chloride (TTC) to make the read easier (ASM). TTC is colorless in its oxidized form. Growing bacteria reduce it to an insoluble red formazan, so growth appears red, and motility appears as small to very large regions of color around the inoculation area. The CDC job aid describes the same picture: red at the inoculation line with pink diffusing outward when the organism is motile.
Direct observation versus the tube
The CDC job aid describes two ways to test motility: a wet mount (direct observation) and motility media. A wet mount lets the observer tell directional motility from Brownian motion. If a wet mount is negative, the job aid says to repeat with a tube test or to incubate a broth. ASM explains why the tube was developed: hanging-drop microscopy can overlook a small number of motile cells among non-motile ones, and motility “may exhibit a cumulative time effect,” so a macroscopic approach was sought.
Reference formulations compared
Two reference sources give a formula for the medium. They share the same base and differ in what is added. Every cell below comes from the named source (ASM protocol, 2016; FDA Bacteriological Analytical Manual, media M103).
| Feature | ASM Motility Test Medium Protocol | FDA BAM Media M103 (semisolid) |
|---|---|---|
| Beef extract | 3.0 g per liter | 3 g per liter |
| Peptone component | Pancreatic digest of casein, 10.0 g per liter | Peptone or gelysate, 10 g per liter |
| Sodium chloride | 5.0 g per liter | 5 g per liter |
| Agar | 4.0 g per liter | 4 g per liter |
| TTC indicator | 5 ml of 1% TTC added | Not listed in the formula |
| Sterilization | Autoclave at 121 C for 15 minutes | Autoclave 15 minutes at 121 C |
| Final pH | Not listed | 7.4 plus or minus 0.2 |
| Incubation stated | 35 C for 18 hours or until growth is evident | No reading criteria given on the page |
| Variants noted | None stated | Higher NaCl (2-3%) for halophilic Vibrio; screw-capped Listeria tubes sealed with parafilm and refrigerated up to two weeks; Salmonella variant poured as plates and used the same day as prepared |
The comparison shows that “motility medium” is best read as a family: an identical nutrient and agar base, with indicator and handling details that depend on the organism group and the method behind it. A medium bought or prepared for one method is not automatically the medium another method names.
Where it matters for labs, clinics and supply decisions
- Match the medium to the method. ASM’s recipe includes TTC; the FDA BAM formula does not list it, and the FDA page documents organism-specific variants (extra salt for halophilic Vibrio, plates for Salmonella). Check which reference a protocol cites before treating two media as interchangeable.
- TTC is not neutral for every organism. The CDC job aid notes that “some organisms are inhibited by the TTC.” A color-based read on a TTC medium therefore depends on the organism tolerating the indicator.
- Temperature is part of the result. ASM cites a 1934 motility study showing that motility varies with incubation temperature, and the CDC job aid refers to incubating at 35 C or the temperature suited to the organism, for 18 to 24 hours. CASRAI’s listeriosis Gram stain guide lists motility test medium among the bench items for that workup.
- Storage and shelf life differ by format. The FDA page states that the Salmonella plate variant should be used the same day it is prepared, while Listeria tubes, screw-capped and sealed, may be refrigerated up to two weeks. Format affects how far ahead a lab can prepare.
- Regulatory class. Under 21 CFR 866.2320 (eCFR, version dated 2026-10-01), a differential culture medium is a Class I device subject to general controls and exempt from premarket notification, subject to the limitations in 866.9. The section does not name motility media specifically, so treat this as the class of differential culture media in general.
Common misunderstandings
“A negative wet mount rules out motility.”
Not by itself. The CDC job aid says to repeat with a tube test or to incubate a broth when a wet mount is negative, and ASM describes hanging-drop observation overlooking a small number of motile cells among non-motile ones.
“Any spread of color means the organism is motile.”
The sources define a positive as diffuse growth outward from the inoculation line or turbidity of the medium. Growth confined to the line is negative. Read the pattern against the reference method rather than the presence of color alone, and remember that TTC can inhibit some organisms.
“Motility agar and motility test medium are different products.”
The sources use “motility test medium” and “motility media” for the semisolid agar; the shorter “motility agar” refers to the same type of preparation. What does differ between preparations is the add-ons described above.
Frequently asked questions
What is motility agar?
It is a semisolid agar medium, a low concentration of agar, used to see whether microorganisms move through the medium (ASM; CDC REACH).
What does a positive result look like?
Diffuse growth outward from the line of inoculation, or turbidity of the medium. With TTC, the CDC job aid describes red at the line and pink diffusing outward.
Why is TTC added?
TTC is reduced by growing bacteria to a red formazan, so growth and its spread become visible as color (ASM). Some organisms are inhibited by it (CDC REACH).
How is it different from a wet mount?
A wet mount observes cells directly and distinguishes directional motility from Brownian motion; the tube medium reads motility macroscopically over incubation time (CDC REACH; ASM).
Related reading: what is bacteriology, what is microbiology, Gram stain reagents and results and what an autoclave does.
Sources
- American Society for Microbiology, Motility Test Medium Protocol (Shields and Cathcart, 2016). Checked 2026-10-31.
- CDC REACH, Core Microbiology Skills: How to Perform a Motility Test (job-aid transcript). Checked 2026-10-31.
- U.S. FDA, BAM Media M103: Motility Test Medium (Semisolid). Checked 2026-10-31.
- eCFR, 21 CFR 866.2320, Differential culture medium. Checked 2026-10-31.
