Growing, culturing and staining microorganisms – Microbiology and Biotechnology (Senior 4 Biology) | Ganzaa

Microbiology and Biotechnology

Lessons

Senior 4 · Biology

Microbiology and Biotechnology

5 lessons

Lesson 3 of 5

Growing, culturing and staining microorganisms

25 min

Why does milk go sour?

Fresh milk left in a jerrycan on a warm afternoon turns sour and thick within a day; the same milk kept in a refrigerator stays fresh for several days; and boiled milk, sealed while hot, lasts longer still. Nothing was added in any case. The difference is in the conditions offered to the bacteria already present in the milk: temperature, food, water and oxygen. Families making ikivuguto use the same knowledge the other way round, keeping milk warm on purpose so that the right bacteria grow.

A microbiologist controls these conditions precisely, to grow one chosen microbe and nothing else. This lesson explains what microbes need to grow, how they are cultured safely in the laboratory, how bacteria are stained to tell them apart, and why viruses need a completely different approach.

Definition

Culture: microorganisms grown in the laboratory on or in a culture medium, a mixture of nutrients that may be liquid (a broth) or solid (set with agar, a jelly from seaweed that most microbes cannot digest).

Colony: a visible mass of cells on a solid medium, all descended from a single cell. A pure culture contains only one species.

Aseptic technique: the set of practices that prevents unwanted microbes from contaminating a culture, the worker or the surroundings.

Sterilisation: killing or removing all microorganisms, including spores, for example in an autoclave (steam at 121 °C under pressure for at least 15 minutes). Disinfection: reducing the number of microbes on objects or surfaces with chemicals.

Obligate aerobe: needs oxygen. Obligate anaerobe: killed or inhibited by oxygen. Facultative anaerobe: grows with or without oxygen.

Plaque: a clear zone in a lawn of bacteria where viruses (bacteriophages) have destroyed the cells.

What microorganisms need to grow

Requirement	Details	Example
Nutrients	A carbon source (sugars, or CO₂ for autotrophs), a nitrogen source (amino acids, ammonium or nitrate), minerals (phosphate, sulfate, potassium, iron) and sometimes growth factors such as vitamins	Nutrient agar contains meat extract, peptone and salt
Water	Needed for enzymes and transport; high salt or sugar draws water out of cells by osmosis	Salted fish and jam resist spoilage
Temperature	Each species has a minimum, optimum and maximum. Psychrophiles grow best below 15 °C, mesophiles at about 20–45 °C (most human pathogens near 37 °C), thermophiles above 45 °C	A refrigerator slows mesophiles in milk
pH	Most bacteria grow best near neutral (pH 6.5–7.5); fungi tolerate more acid (pH 4–6)	Acid in ikivuguto and pickles stops many spoilage bacteria
Oxygen	Requirements range from essential to toxic (Figure 1)	Tetanus bacteria grow in deep, airless wounds

Culture media

Type	What it does	Example
General-purpose	Supports a wide range of microbes	Nutrient agar, nutrient broth
Enriched	Extra nutrients for fussy (fastidious) organisms	Blood agar
Selective	Contains substances that stop some microbes and let the wanted ones grow	MacConkey agar: bile salts and crystal violet inhibit many Gram-positive bacteria
Differential	Makes different microbes look different	MacConkey agar: lactose fermenters such as E. coli form pink colonies

Aseptic technique: a streak plate

A streak plate thins out a culture across the agar until single cells are left far apart; each grows into a separate colony, which can be picked to start a pure culture.

Wash your hands, tie back hair and disinfect the bench with a disinfectant such as 70 % ethanol (keep it away from the flame).
Work close to a lit Bunsen burner: its rising hot air carries airborne microbes upwards, away from the plate.
Heat the inoculating loop until it glows red-hot along its whole length, then let it cool for a few seconds near the flame (a hot loop kills the bacteria you want to transfer).
Open the culture bottle, pass its neck through the flame, take a loopful, flame the neck again and close it.
Lift the Petri dish lid only slightly, like a shield, streak as in Figure 2, and close it at once.
Re-flame the loop. Tape the lid with two short strips (not all round, so that air can enter and anaerobes are not encouraged), label the base, and incubate upside down so condensation does not drip onto the agar.

A streaked agar plate. Where the loop first spread the culture (left) the streaks have grown into solid lines; along the last streaks (right) the cells were far enough apart to grow into separate round colonies, each a clone of one cell. Photo: public domain, via Wikimedia Commons

Warning

Biosafety in the school laboratory

Use only cultures supplied for teaching (harmless strains). Never culture from people, toilets, raw meat, soil from latrines or drains.
Incubate at no more than 30 °C (25 °C is ideal). Body temperature, 37 °C, favours the growth of human pathogens.
Once a plate is taped and incubated, never open it: look through the lid. Mixed cultures can contain pathogens even if you started with a harmless strain.
Do not eat, drink or touch your face in the laboratory; wash your hands before and after practical work.
After use, all cultures and plates are sterilised in an autoclave or pressure cooker (121 °C) by the teacher before disposal. Spills are covered with disinfectant, left for 15 minutes, then cleared up with gloves.

Staining bacteria: the Gram stain

Bacterial cells are almost transparent, so they are stained before viewing. The Gram stain (S4 Unit 2) sorts bacteria into two groups that differ in cell wall structure and often in which antibiotics affect them.

Step	Reagent and time	Why
1. Fix	Thin smear dried and passed through a flame	Sticks cells to the slide and kills them
2. Primary stain	Crystal violet, about 1 minute	Stains all cells purple
3. Mordant	Iodine, about 1 minute	Forms a large crystal violet–iodine complex in the cells
4. Decolourise	Alcohol or acetone, a few seconds only	Washes the complex out of Gram-negative cells (thin peptidoglycan, outer membrane dissolved) but not Gram-positive cells (thick peptidoglycan traps it)
5. Counterstain	Safranin, about 1 minute	Stains the now colourless Gram-negative cells pink

Result: Gram-positive cells are purple, Gram-negative cells pink. Decolourising for too long, or using an old culture whose walls are breaking down, can make Gram-positive cells look pink. Other stains reveal other features: the acid-fast (Ziehl–Neelsen) stain for Mycobacterium, spore stains for endospores, and negative stains that colour the background to show capsules.

Growing viruses

Viruses multiply only inside living cells, so they cannot be grown on nutrient agar. Three hosts are used:

Bacteria, for bacteriophages: phage and bacteria are spread together on agar; the bacteria form a cloudy lawn, and each phage that infects a cell starts a cycle of infection and bursting that leaves a clear plaque.
Embryonated (fertilised) chicken eggs: virus injected into the fluids or membranes of the developing embryo multiplies there; most influenza vaccines are still made this way.
Cell (tissue) cultures: animal or plant cells grown in sterile flasks; virus growth shows as damage to the cells (a cytopathic effect).

Worked example 1: what kind of microbe is it?

A bacterium was grown at different temperatures and in tubes like those in Figure 1 (illustrative results). Growth after 2 days: 10 °C, none; 25 °C, good; 37 °C, best; 50 °C, none. In the tube, growth was densest at the top but present all the way down. Classify the bacterium and suggest where it might live.

Temperature: it grows between about 25 °C and 37 °C, best at 37 °C, and not at 50 °C, so it is a mesophile with an optimum near body temperature.
Oxygen: growth throughout the tube, densest at the top, means it can grow without oxygen but grows better with it (aerobic respiration releases more energy): a facultative anaerobe.
A mesophilic facultative anaerobe with an optimum of 37 °C fits a bacterium of the human or animal gut, such as E. coli; this is exactly why school cultures must not be incubated at 37 °C.

Worked example 2: counting viruses with a plaque assay

A phage suspension was diluted to $10^{-6}$ of its original concentration, and 0.1 cm³ of the dilution was mixed with bacteria and spread on a plate. After incubation there were 45 plaques. Calculate the number of plaque-forming units (PFU) per cm³ in the original suspension.

Each plaque started from one infective phage, so the 0.1 cm³ of diluted suspension held 45 PFU.
$\text{PFU per cm}^3 = \frac{\text{number of plaques}}{\text{volume plated} \times \text{dilution}} = \frac{45}{0.1 \times 10^{-6}} = 4.5 \times 10^{8}\ \text{PFU cm}^{-3}$
Plates with about 30 to 300 plaques are used: fewer gives an unreliable count, more and the plaques overlap.

Microbiology at the University of Lincoln: "Streak Plating". Watch how the loop is sterilised, how little the lid is lifted, and how the loop is flamed between each set of streaks.

Tip

Memory hook. What microbes need: "Warm Food, Water, PH and Oxygen" (W-F-W-P-O, "Who Feeds Wild Pigs Oats?"). Gram stain order: "Come In And Sit": Crystal violet, Iodine, Alcohol, Safranin.

✦

Practice: culturing microorganisms

List five requirements for the growth of bacteria and give one way each is used to preserve food.
Explain why agar is used to make solid culture media.
Distinguish between selective and differential media, using MacConkey agar as an example.
Explain the purpose of each of these: (a) flaming the loop until red-hot, (b) working beside a Bunsen flame, (c) incubating plates upside down, (d) not sealing the lid all the way round.
Explain why Gram-positive bacteria stay purple after the alcohol step, while Gram-negative bacteria lose the purple colour.
Explain why viruses cannot be grown on nutrient agar, and name two ways of growing them.
A phage suspension was diluted to $10^{-5}$ ; 0.1 cm³ of this dilution gave 38 plaques. Calculate the PFU per cm³ in the original suspension.

Show answersHide answers

Nutrients, water, a suitable temperature, a suitable pH and the right oxygen supply. Preservation: remove water (drying fish, salting, adding sugar to jam); low temperature (refrigerating); acid pH (pickling, fermented milk); remove oxygen (sealed cans, vacuum packs); nutrients cannot easily be removed, so sterilise instead (boiling, canning).
Agar is a seaweed jelly that melts near 85 °C and sets as it cools below about 40 °C, so it can be poured hot and is solid at incubation temperatures; most microbes cannot digest it, so the surface stays firm; it lets colonies grow separately so they can be counted and picked.
A selective medium inhibits unwanted microbes and lets others grow: MacConkey's bile salts and crystal violet inhibit many Gram-positive bacteria. A differential medium makes different microbes look different: on MacConkey, lactose fermenters form pink colonies and non-fermenters pale ones. MacConkey is both.
(a) Kills all microbes on the loop, so only the intended culture is transferred. (b) The rising hot air carries airborne microbes upwards, away from the open plate. (c) Stops condensation dripping onto the agar and spreading colonies together. (d) Lets air in, so anaerobes, which include many pathogens, are not encouraged.
Gram-positive cells have a thick peptidoglycan wall that traps the large crystal violet–iodine complex when the alcohol dehydrates it, so they stay purple. Gram-negative cells have a thin peptidoglycan layer and an outer membrane that alcohol dissolves, so the complex washes out; they are later stained pink by safranin.
Viruses have no metabolism or ribosomes and can multiply only inside living host cells, which nutrient agar does not contain. They are grown in bacteria (phages, as plaques on a lawn), in embryonated chicken eggs, or in animal or plant cell cultures.
$\frac{38}{0.1 \times 10^{-5}} = 3.8 \times 10^{7}$ PFU per cm³.

Quick check

A bacterium grows only in the bottom of a tube in which oxygen is plentiful at the top. What is it?

Key points

Microbes need nutrients, water, a suitable temperature (psychrophile, mesophile, thermophile), pH and oxygen supply (obligate aerobe, facultative, microaerophile, obligate anaerobe).
Media can be general, enriched, selective or differential; agar makes them solid.
Aseptic technique (flamed loop, Bunsen updraught, lid barely lifted, taped not sealed, incubate at 30 °C or below, autoclave everything after) protects the culture, the worker and the surroundings.
Gram stain: crystal violet, iodine, alcohol, safranin; positive = purple (thick peptidoglycan), negative = pink.
Viruses are grown in bacteria (plaques), eggs or cell cultures; PFU per cm³ = plaques ÷ (volume × dilution).

Sources and credits

9.2: Oxygen Requirements for Microbial Growth, 9.4: Temperature and Microbial Growth, 9.6: Media Used for Bacterial Growth, 13.2: Using Physical Methods to Control Microorganisms and 6.3: Isolation, Culture, and Identification of Viruses, from Microbiology by OpenStax (Nina Parker, Mark Schneegurt, Anh-Hue Thi Tu, Philip Lister, Brian M. Forster), via LibreTexts Biology, licensed under CC BY 4.0. Adapted by Ganzaa.
Figures 1 and 2, the tables, worked examples (the temperature results are illustrative), practice and quick check: written by Ganzaa. Laboratory safety rules follow common school microbiology practice.
Photos: public domain (agar plate); Irhode01, CC BY-SA 4.0 (phage plaques); both via Wikimedia Commons.
Video: "Streak Plating", Microbiology at the University of Lincoln, embedded from YouTube.

1Why does milk go sour?

2What microorganisms need to grow

3Culture media

4Aseptic technique: a streak plate

5Staining bacteria: the Gram stain

6Growing viruses