This media is differential. It evaluates an organism’s capacity to convert glucose and lactose into acids and acids plus gases. It also makes it possible to identify sulfur reducers. This medium is frequently used to distinguish between members of the Enterobacteriaceae family that ferment lactose, such as Escherichia coli, and those that do not, such as Shigella dysenteriae. These lactose nonfermenting enterics are typically the more dangerous gastrointestinal pathogens.
Glucose, the primary differentiating component, is quite scarce. This sugar will be consumed within the first few hours of incubation by organisms that can ferment it. Acidic byproducts produced during glucose fermentation will cause the media’s phenol red indicator to turn yellow. The tube will therefore be fully yellow following the first few hours of incubation. The organism now needs to choose a different food source because all of the available glucose has been consumed. This is the sugar the organism will pick if it can ferment lactose. The media will continue to be yellow and lactose fermentation will continue to produce acidic byproducts (picture on the far left below). Fissures will form in the agar or the agar will lift off the tube’s bottom if gas is created as a result of the fermentation of glucose or lactose.
An organism will have to utilise the amino acids and proteins in the medium if it cannot use lactose as a food source. The medium turns alkaline as a result of the amino acids being converted to NH3, a weak base. The phenol red indicator starts to glow red due to the alkaline pH. Only the slant of the tube has a chance to turn red because the incubation period is so brief (1824 h). In a KIA tube, a bacterium that can only ferment glucose but not lactose will consequently create a red slant and a yellow butt (second from the left below). These are the GIT’s more dangerous pathogens, like Shigella dysenteriae.
An organism will only use amino acids or proteins if it is incapable of utilizing glucose or lactose. The butt’s color will not change, and the tube’s slant will be red (picture on the far right below). A nonfermenter is an example of Pseudomonas aeruginosa.
The generation of H2S can also be detected by KIA tubes. It appears to be a dark precipitation (second picture from the right). The butt of the tube can occasionally be obscured by the dark precipitation. When this occurs, the organisms should be regarded as showing signs of glucose fermentation (yellow butt). Proteus mirabilis is a glucose positive, lactose negative, sulfur reducing enteric (seen below, second from right).
In This Article...
Which carbs are measured by the KIA?
You must keep in mind that this test is called “Triple Sugar Iron Agar” and that it comprises agar as a solidifying agent together with three sugars (lactose, sucrose, and glucose) (TSI is a semi-solid media having slant and butt).
If the color of the KIA slant changes, what does that tell us?
An indication of fermentation of carbohydrates is phenol red. On the slant and in the butt, fermentation reactions are detected by a color change from red (alkaline) to yellow (acid). The amount of dextrose in KIA is a tenth of the amount of lactose.
Why does TSI Agar contain more lactose and sucrose than glucose?
Since lactose and sucrose are present in much higher concentrations than glucose, acid formation in the butt is caused by these sugars, whereas glucose acid formation is suppressed by a rapid oxidation of the small amount of acid in the slanted area of the tube, resulting in a neutral or alkaline pH reaction when the reaction is completed.
Carbohydrate Fermentation:
- Slant Reaction Positive Test Yellow (acid)
- Slant Reaction Red Test Results: Negative (alkaline)
- Positive Butt Reaction TestYellow (acid)
- Butt ReactionRed Test Results: Negative (alkaline)
Hydrogen Sulfide Production:
- Result of Test A black precipitate in the butt, a black ring at the intersection of the slant and butt, or a black tint throughout the medium
- Unfavorable Test
- no development of the color black
Gas Production:
- Positive TestBubbles in the medium, medium displacement or breaking, or medium separation from the tube’s side or bottom
- Unfavorable Test
- There are no bubbles, and the medium has not separated or moved.
What do the KIA test results mean?
A differential slope is called Kligler iron agar (KIA).
utilized as a tool for identifying Salmonella, Shigella, and other active bacteria.
The basis for KIA reactions is the fermentation of
Composition of KIA:
- KIA is prepared from an economic and practical standpoint.
- Powder from Lab-Lemco
- ……………………………………..3.0 gm/ltr
- Peptone…………………………………………..20.0 gm/ltr
- Chloride of sodium
- ………………………………………….5.0 gm/ltr
- Yeast extract………………………………………..3.0 gm/ltr
- Lactose…………………………………………………10.0 gm/ltr
- Dextrose…………………………………………………..1.0 gm/ltr
- Iron citrate
- …………………………………………….0.3 gm/ltr
- 0.3 gm/ltr of sodium thiosulphate is the recommended amount.
- Phenol red……………………………………0.05 gm/ltr
- Agar. 12.0 gm/ltr………………………………………………………………………
Preparation of KIA:
- 5.5 grams of the medium are used for every 100 ml of distilled water.
- As directed by the manufacturer, prepare.
- Mix thoroughly and pour 6 ml portions into big size tubes once the mixture has cooled to 5055C.
- By autoclaving (with the caps off) at 121C for 15 minutes, sterilize.
- Allow the medium to firm in a sloping position to produce a butt and slope that are each about 25 mm long and 25 mm deep.
- Give the medium a batch number and a date.
Use of KIA:
- used to distinguish between Salmonella, Shigella, and other enteria bacteria based on the generation of H2S gas and the fermentation of glucose and lactose.
- *Inject KIA medium with a straight wire by stabbing it in the butt first and then zigzagging across the slope. Make sure the tube tops are kept unfastened after vaccination.
Result interpretation of triple sugar iron test on KIA
- Only glucose is fermented, as indicated by the hot pink slope and yellow butt (acid generation). Because of a reverse ion of the acid reaction occurring in aerobic conditions, the slope appears pink-red. Salmonella, Shigella, and other intestinal infections can cause this reaction.
- The presence of bubbles and cracks in the medium indicates fermentation of glucose has produced gas. Salmonella paratyphi and several faecal commensals create gas.
- Lactose and maybe glucose are fermented when there is a yellow butt and slope. E. coli and other enterobacteria do this.
- Lack of lactose or glucose fermentation is indicated by a hot pink slope and butt. Most Pseudomonas aeruginosa strains exhibit this.
- Hydrogen sulfide (H2S) generation is indicated by blackening along the stab line or across the medium.
Reversion occurs in phenol red broth for what reason?
An organic molecule donates electrons during the metabolic process, and one or more of its organic compounds serves as the final electron acceptor.
What major group of bacteria may be distinguished most effectively using carbohydrate fermentation tests?
Why is it not advised to read a phenol red broth test after 18 hours of incubation?
Because there is a chance of a reversion, which occurs when an organism flips from the metabolic state of fermentation to that of deamination after using all available sources of glucose. A true non-fermenter cannot be visually distinguished from a reversion.
When an organism reverses metabolic modes, from fermentation to deamination of peptone amino acids, AFTER the exhaustion of carbohydrates, the medium undergoes a reversion.
What element of the medium has been fermented, as indicated by the phenol red broth’s yellow color?
What medium component has been catabolized, as seen by the pink color of the phenol red broth?
Why is it crucial to distinguish between Enterobacteriaceae and glucose Nonfermenters?
Why is it crucial to distinguish between Enterobacteriaceae and glucose nonfermenters? -since nonfermenters exhibit higher levels of common antibacterial agent resistance.
General
When you see the name of this test, Triple Sugar Iron Agar, you must keep in mind that it is a test that contains iron as well as three sugars (Lactose, Sucrose, and Glucose), and it also contains agar as a solidifying agent (TSI is a semi-solid media having slant and butt).
10:10:1 (i.e., 10 parts Lactose (1%) and 10 parts Sucrose (1%) and 1 part Glucose (0.1%)) is the ratio of the sugars lactose, sucrose, and glucose.
– 0.1 percent Glucose: When glucose is the only ingredient fermented, only enough acid is created to tint the butt yellow. The red slant will continue.
-1.0% lactose/1.0% sucrose: If lactose, sucrose, or both sugars are fermented, a significant amount of acid is produced, turning both the butt and slant yellow. This means that the isolate can ferment lactose, sucrose, or both, as seen by the emergence of yellow color in both slant and butt.
– Phenol red: Acidification indicator (It is yellow in acidic condition and red under alkaline conditions).
– Peptone, which serves as a source of nitrogen, is also present. (Keep in mind that ammonia is created whenever peptone is used in an aerobic environment.)
When sucrose is added to TSI Agar, coliform bacteria that ferment sucrose more quickly than lactose can be found early. Additionally, the addition of sucrose helps identify certain gram-negative bacteria that can ferment sucrose but not lactose. Another fundamental knowledge is that TSI tubes have a butt (a poorly oxygenated portion on the bottom) tilt (angled well-oxygenated area on the top).
1) Inoculate a well-isolated colony by touching the top with a sterilized straight inoculation needle.
2) Streak over the surface of the agar slant after streaking through the center of the medium to the bottom of the tube to inoculate TSI Agar.
3) Place the cap loosely back on and incubate the tube for 18 to 24 hours at 35C in room-temperature air.
1) Lactose (or sucrose) fermentation results in the production of a significant amount of acid, which causes the phenol red indicator to turn yellow both in the butt and in the slant. Some organisms release gases, which cause the medium to bubble or break.
2) If lactose is not fermented but a tiny amount of glucose is, the oxygen-deficient butt will be yellow (keep in mind that butt relatively have more glucose than slant, i.e., more media more glucose), but on the slant the acid (less acid as media in slant is very little) will be oxidized to carbon dioxide and water by the organism and the slant will be red) (alkaline or neutral pH).
3) The butt and the slant will both be crimson if neither lactose/sucrose nor glucose is fermented. The formation of ammonia from the oxidative deamination of amino acids might cause the slant to turn a deeper red-purple (more alkaline) (remember peoptone is a major constituent of TSI Agar).
4) The presence of ferrous sulfide, which is black, indicates the presence of H2S.
History
Sulkin and Willett first disclosed a medium in 1917 that contained iron salts together with the sugars glucose, lactose, and sucrose.
These carbohydrates were fermented in the medium, and hydrogen sulfide was also produced.
The pH indicator was added to the medium by Hajna in 1945, and this version is still in use today.