Microbiology Lab Practical Terms

Aseptic technique - definition

Practices that keep unwanted microbes out of cultures and media and keep lab organisms contained: keep lids closed, never touch insides of plates/tubes/lids, hold caps in hand, flame loops/needles (10 sec) and tube necks (3 sec), incubate plates upside down

Aseptic technique - main purpose

Prevent contamination so cultures stay pure, and contain microorganisms to protect you and others (contamination is very easy)

Why sterilize the loop or needle before a transfer?

To kill any microbes on it so only the intended organism is transferred and the culture/media are not contaminated (10 sec in micro-incinerator, then cool 10-15 sec so it does not kill the bacteria)

What goes in the biohazard bag?

Used/old cultures (plates and unbroken tubes), stock cultures, gloves, and Spor-Klenz paper towels. (Broken tubes go in the tan bin over the mini fridge; slides go in the red sharps bin)

Microscope part that moves the stage side to side

Stage controls (mechanical stage controls)

Microscope part that moves the stage up and down

Coarse and fine adjustment knobs

Shape of the Gram-positive bacteria in lab (S. epidermidis)

Cocci (round), purple. Note: cocci are not always Gram-positive

Shape of the Gram-negative bacteria in lab (E. coli)

Bacilli (rods), reddish-pink. Note: bacilli are not always Gram-negative

Coccus vs bacillus

Coccus = round; bacillus = rod. (Vibrio = curved rod; spirochete = spiral)

Simple stain - primary function

Colors the whole cell to add contrast so you can see cell shape (morphology), arrangement (clusters, chains, pairs) and size. It is non-differential

Simple stain - charge of the stain

Positively charged chromogen; binds the negatively charged bacterial cell surface, so cells take the stain color

Why are unstained cells hard to see in bright-field microscopy?

Bacterial cells are colorless/transparent, so there is almost no contrast with the background

Simple stain step that shrinks cytoplasm and makes cells observable

Heat fixing (10 sec, marker side against the micro-incinerator). Longer can lyse the cells

Negative stain - key facts

Nigrosin (negative chromogen) colors the background, cells stay white; NOT heat fixed, so cells keep true size and shape

Gram stain procedure

1) Mixed smear, air dry, heat fix 10 sec 2) Crystal violet (primary stain) 60 sec, DI rinse 3) Gram's iodine (mordant) 60 sec, rinse 4) Decolorizer (alcohol) count to 3, rinse immediately 5) Safranin (counterstain) 60 sec, rinse 6) Blot dry with bibulous paper

Gram stain - role of crystal violet

Primary stain; stains all cells purple

Gram stain - role of iodine

Mordant; locks the crystal violet into the cell

Gram stain - role of decolorizer

Alcohol removes crystal violet from Gram-negative cells (thin peptidoglycan) but not Gram-positive cells

Gram stain - role of safranin

Counterstain; turns the decolorized Gram-negative cells reddish-pink

Why is Gram staining usually the first test on an unknown?

It is the most important differential stain: it quickly splits bacteria into two groups by cell wall structure, which narrows the identity and decides which tests come next (first step of every flowchart; guides treatment in medicine)

Gram-positive color

Purple (thick peptidoglycan, no outer membrane, resists decolorization)

Gram-negative color

Reddish-pink (thin peptidoglycan plus lipopolysaccharide outer membrane, decolorized by alcohol, picks up safranin)

Over-decolorizing a Gram stain causes

Gram-positive cells appear reddish (false Gram-negative)

Under-decolorizing a Gram stain causes

Gram-negative cells appear purple (false Gram-positive)

General-purpose media

Allows growth of most heterotrophic organisms. Examples: TSA plates, TSB broth, nutrient broth, nutrient agar

Selective media

Inhibits growth of some species while encouraging (selecting for) growth of others

Main question answered by selective media

Who is allowed to grow?

How does selective media prevent certain organisms from growing?

It contains inhibitory agents: antibiotics (colistin and nalidixic acid in CNA), high salt (7.5% NaCl in MSA), bile salts and crystal violet (MacConkey), eosin and methylene blue dyes (EMB)

Differential media

Distinguishes between species by their growth forms or biochemical activities, usually shown by a visible color/indicator change

Main question answered by differential media

What does this bacterium do once it grows here?

What characteristics can differential media detect?

Sugar fermentation (acid and/or gas), hemolysis, enzyme production (catalase, oxidase, DNase, urease, deaminase, decarboxylase), H2S production, indole production, motility, citrate use

Enriched media

Contains added nutrients or growth factors; asks who needs extra nutrients to grow (e.g., sheep's blood in CNA)

Can one medium be both selective and differential?

Yes. CNA, MSA, MacConkey and EMB are all both. Example: MSA's salt selects for Gram-positive halophiles, while mannitol plus phenol red differentiates mannitol fermenters

Why is carbohydrate metabolism useful for identifying bacteria?

Each species uses a specific set of sugars and reactions, and the end products (acid, gas) differ, so the pattern of which sugars are fermented helps tell species apart

How is acid production detected in media?

A pH indicator changes color as pH drops (phenol red: red to yellow; neutral red on MAC: pink; methyl red: red)

How is gas production detected?

A bubble in the inverted Durham tube (broths); bubbles or cracked/split agar (TSI)

What happens to pH when acidic fermentation products accumulate?

pH decreases (becomes more acidic), which changes the indicator color

CNA - selective, differential, or both?

Both

CNA - what group is selected for?

Gram-positive bacteria

CNA - what group is inhibited and by what?

Gram-negative bacteria, by the antibiotics colistin and nalidixic acid

CNA - what activity is differentiated?

Hemolysis (breakdown of the red blood cells in sheep's blood)

Alpha hemolysis

Incomplete hemolysis; greenish halo around the colony

Beta hemolysis

Complete hemolysis; clear zone around the colony

Gamma hemolysis

No hemolysis; no change to the medium

Which hemolysis is complete destruction of RBCs?

Beta

Which hemolysis is incomplete?

Alpha

Which hemolysis is none?

Gamma

MSA - selective, differential, or both?

Both

MSA - selective component

7.5% sodium chloride (high salt)

MSA - organisms selected by high salt

Gram-positive halophiles (salt-tolerant), such as staphylococci; Gram-negatives are inhibited

MSA - differential carbohydrate

Mannitol

MSA - metabolic reaction tested

Mannitol fermentation

MSA - pH when mannitol is fermented

Decreases (acidic byproduct)

MSA - color change for mannitol fermentation

Red to yellow (phenol red indicator)

MSA - growth but no yellow color means

Organism tolerates salt but does not ferment mannitol (mannitol negative), e.g., S. epidermidis

MSA - no growth: can you conclude it cannot ferment mannitol?

No. The salt inhibited it, so fermentation was never tested (e.g., E. faecalis can ferment mannitol but grows little or not at all on MSA)

MacConkey - selective, differential, or both?

Both

MacConkey - organisms selected for

Gram-negative bacteria

MacConkey - organisms inhibited

Gram-positive bacteria (by bile salts and crystal violet)

MacConkey - carbohydrate tested

Lactose

MacConkey - biochemical reaction tested

Lactose fermentation

MacConkey - pH when lactose is fermented

Decreases (acid byproduct)

MacConkey - color of lactose fermentation

Pinkish-red/pink colonies (neutral red indicator); non-fermenters stay pale/colorless

MacConkey - no growth suggests

The organism is Gram-positive (inhibited by bile salts and crystal violet)

EMB - selective, differential, or both?

Both

EMB - group selected for

Gram-negative bacteria

EMB - group inhibited

Gram-positive bacteria (by eosin and methylene blue dyes)

EMB - carbohydrate tested

Lactose

EMB - metabolic activity differentiated

Lactose fermentation (fermenters vs non-fermenters, and how strongly)

EMB - dark colony indicates

Lactose fermentation; the acid makes eosin and methylene blue combine into a dark purple precipitate

EMB - metallic green sheen suggests

Strong/vigorous lactose fermentation producing lots of acid (classic E. coli)

EMB - lactose non-fermenter vs strong fermenter

Non-fermenter: colorless/pale colonies with no dark precipitate. Strong fermenter: dark purple-black colonies, often with a metallic green sheen

EMB vs MacConkey - similarity

Both select for Gram-negatives (inhibit Gram-positives) and differentiate lactose fermenters from non-fermenters

EMB vs MacConkey - indicator difference

MacConkey uses neutral red pH indicator (fermenters turn pink); EMB uses eosin and methylene blue dyes that form a dark purple/metallic green precipitate

Phenol red broth - selective or differential?

Differential (each tube has one sugar: glucose green cap, lactose blue cap, sucrose red cap, plus pH indicator and Durham tube)

Phenol red broth - yellow means

Acid produced; the organism fermented that sugar (positive)

Phenol red broth - what causes the color change?

Acidic byproducts of carbohydrate fermentation lower the pH

Phenol red broth - red/pink means

No fermentation of that sugar (negative)

Phenol red broth - gas production shown by

A bubble trapped in the Durham tube

Methyl red test - purpose

Identifies organisms that perform mixed acid fermentation of glucose and produce stable acid end products

Methyl red - pH when stable acids accumulate

Stays low; the broth's pH is lowered permanently

Methyl red - true positive color

Red (after adding 3 drops methyl red; read immediately). Yellow/orange = negative

Methyl red positive tells you

The organism ferments glucose by the mixed-acid pathway, making enough stable acid to keep the pH low

Simmons citrate - only carbon source

Sodium citrate

Why is citrate as the sole carbon source important?

The organism can only grow if it can take up and use citrate, so growth/color change proves citrate utilization

Citrate - positive color

Blue

Citrate - negative color

Green (no change)

Citrate positive tells you

The organism makes citrate permease and can use citrate as its only carbon source

TSI - overall purpose

Differentiates glucose fermenters from lactose/sucrose fermenters, and detects gas and H2S production (stab and streak with needle)

TSI - three carbohydrates

Glucose (0.1%), lactose (1%), sucrose (1%)

TSI - yellow means

Acid from carbohydrate fermentation

TSI - red means

No fermentation (alkaline/no acid)

TSI - yellow slant/yellow butt

Ferments glucose plus lactose and/or sucrose (acid/acid)

TSI - red slant/red butt

Does not ferment any of the three sugars (non-fermenter)

TSI - red slant/yellow butt

Ferments glucose only (not lactose or sucrose)

TSI - color for H2S production

Black precipitate

TSI - gas production

Bubbles or split/cracked agar

Catalase test - purpose

Detects the enzyme catalase, which breaks down toxic hydrogen peroxide (H2O2); common in Staphylococcus

Catalase - positive

Bubbles when H2O2 is added

Catalase - negative

No bubbles