FOOD MICROBIOLOGY: STAINING AND IDENTIFICATION
GRAM-POSITIVE
Thick peptidoglycan wall (20 to 80 nm), no outer membrane. Holds crystal violet and stays purple.
GRAM-NEGATIVE
Thin peptidoglycan wall (5 to 10 nm) plus an outer membrane with lipopolysaccharide. Loses crystal violet and turns pink with safranin.
Figure 1: The Gram stain separates bacteria by cell wall structure: thick wall holds the purple dye, thin wall with an outer membrane loses it.
Introduction
Gram-positive and Gram-negative bacteria differ in cell wall structure, and the Gram stain tells them apart in about ten minutes. Gram-positive cells have a thick peptidoglycan wall and stay purple, while Gram-negative cells have a thin wall plus an outer membrane and turn pink.
Quick comparison
Quick comparison: Gram-Positive and Gram-Negative Bacteria
| Aspect | Gram-Positive Bacteria | Gram-Negative Bacteria |
|---|---|---|
| Peptidoglycan layer | Thick, 20 to 80 nm | Thin, 5 to 10 nm |
| Outer membrane | Absent | Present, with lipopolysaccharide and lipoprotein |
| Teichoic acid | Present in the wall | Absent |
| Endotoxin (lipopolysaccharide) | Absent | Present in the outer membrane |
| Gram stain result | Purple: holds the crystal violet-iodine complex | Pink or red: loses the complex, coloured by safranin counterstain |
| Lysozyme sensitivity | Wall is digested by lysozyme | Outer membrane gives more protection |
| Food examples | Staphylococcus, Streptococcus, Lactobacillus, Listeria, Bacillus, Clostridium | Escherichia coli, Salmonella, Pseudomonas, Enterobacter, Klebsiella, Campylobacter, Vibrio |
| Use in food | Lactic acid bacteria ferment yogurt, cheese and sausage; Bacillus and Clostridium spores survive heat | Coliforms act as hygiene indicators; Pseudomonas spoils chilled meat and milk |
| First identification step | Gram-positive coccus in clusters points to Staphylococcus; in chains to Streptococcus | Gram-negative rod points to groups such as Escherichia, Salmonella and Pseudomonas |
Scientific explanation
Definition
Gram-positive bacteria have a thick peptidoglycan wall outside the cell membrane and no outer membrane. Gram-negative bacteria have a thin peptidoglycan layer overlaid by an outer membrane whose main molecules are lipopolysaccharides and lipoprotein. The grouping comes from the Gram stain, developed by Hans Christian Gram in 1884.
Working principle
Crystal violet stains all cells purple. Iodine acts as a mordant and forms a large crystal violet-iodine complex. Alcohol decolourizes: the complex is trapped in the thick peptidoglycan of Gram-positive cells, which stay purple, but it is washed out of Gram-negative cells, which become colourless. Safranin then colours the Gram-negative cells pink or red.
Apparatus
Clean slides, a loop, a burner for heat fixing, crystal violet, Gram’s iodine, alcohol decolourizer, safranin, and a light microscope with oil immersion. A known Gram-positive and a known Gram-negative control on the same slide show up a staining error at once.
Measurement procedure
Make a thin smear, air dry, heat fix, flood with crystal violet for one minute, rinse, add Gram’s iodine for one minute, rinse, decolourize with alcohol for a few seconds until the run-off is clear, rinse at once, counterstain with safranin for about one minute, rinse, blot dry, and examine under oil immersion.
Factors affecting readings
Over-decolourizing turns Gram-positive cells pink; under-decolourizing leaves Gram-negative cells purple; old cultures of Gram-positive cells can look Gram-negative because their walls weaken. The stain narrows the field but never names a species by itself.
Formulas and calculations
This topic has no standard formula. The Gram stain is a qualitative test, and its result is a colour and a shape, not a number.
Industrial applications
- Food labs: first sorting step for any isolate from food, guiding the choice of selective media and biochemical tests.
- Fermentation: Gram-positive lactic acid bacteria make yogurt, cheese, pickles and fermented sausage.
- Hygiene testing: Gram-negative coliforms and Pseudomonas are tracked as indicators and spoilage organisms.
Advantages and limitations
Gram-positive: advantages are a simple, fast stain and a clear first sorting step; limitations are that the stain alone cannot name a species, and spore formers such as Bacillus and Clostridium survive heat that kills vegetative cells.
Gram-negative: advantages are that the outer membrane and endotoxin give clear test targets, and coliform testing gives a fast hygiene signal; limitations are that many Gram-negative pathogens carry endotoxin, and the outer membrane can block some dyes and chemicals.
FAQ
What does the Gram stain actually test?
It tests cell wall structure: whether the wall is thick enough to trap the crystal violet-iodine complex during decolourizing.
Why do old Gram-positive cultures sometimes look pink?
Their walls weaken with age, so the dye washes out during decolourizing, and the cells take up the pink counterstain.
Does a Gram stain name the species?
No. It narrows the field to a group, such as Gram-negative rods, and culture on selective media plus biochemical tests finish the identification.
Why does the outer membrane matter in food?
Its lipopolysaccharide is endotoxin, and it controls what enters the cell, which changes how the organism reacts to dyes, chemicals and sanitizers.
References and further reading
FTN subjects used: Food Microbiology (Units 1, 2, 3, 4, 5 and 8) and General Microbiology (Units 1, 2, 3, 4, 5, 6 and 10), from the notes at ~/workspace/food-tech-notes/notes/.
Books used: Adams, M. R. and Moss, M. O., Food Microbiology, Royal Society of Chemistry, 2007 (book on disk at food-tech-notes/books/Food-Microbiology.pdf; Google Books page: https://books.google.com/books?id=BZNKI088iooC). Ray, B. and Bhunia, A., Fundamental Food Microbiology, 5th edition, as cited in the FTN notes. Essentials of Industrial Microbiology (book on disk at food-tech-notes/books/Essentials-of-Industrial-Microbiology.pdf).
Google Books pages used: Food Microbiology by Adams and Moss, https://books.google.com/books?id=BZNKI088iooC (contents and chapter list read on 2026-11-11).
