grams staining and yoghurt making

Investigating prokaryotes

IB Biology

Label this cell!

Cell Wall

• Determine shape of bacteria

• Strength prevents osmotic rupture

• 20-40% of bacteria

• Unique to bacteria

• Some antibiotics effect directly– Penicillin

Cell Wall

• Peptidoglycan polymer -amino acids + sugar

• Amino acids cross link sugars

Gram Staining

• Technique named after Hans Christian Gram (Danish scientist)

• Developed technique in 1884

• Used to differentiate between bacteria that show same clinical symptoms

• Thick peptidoglycan

• 90% peptidoglycan

• Teichoic acids• 1 layer• Not many

polysaccharides

Gram-positive cell walls Gram-negative cell

• Thin peptidoglycan• 5-10% peptidoglycan• No teichoic acids• 3 layers• Outer membrane has

lipids, polysaccharides

The characteristic compound found in all true bacterial cell walls is peptidoglycan.

The amount of peptidoglycycan is among one of the differences between the Gram Positive and Gram Negative cell walls.

Figure 4.13b, c

The process includes the use of:a primary stain (crystal violet) a mordant (helper) iodine solution, a decolorizer (95% ethanol), a counterstain (safranin).

Patient No. 986214

• Admitted at 01.03 this morning after complaining of severe stomach cramps and nausea

• Case history revealed that the patient bought a tamale from a street vendor on his way home from a late shift at the Jumex factory yesterday night

• After feeling okay for 24 hours after consumption of the tamale the cramping began

Your task

• The doctor wants to rule out bacterial food poisoning from Salmonella

• Bacterial cultures from the patient´s blood and stomach contents have been prepared

• Your job as the hospital microbiologist is to test the sample for presence of Salmonella

Salmonella toxins

Enterotoxin:• released by microorganism in the lower intestine• alters permeability of intestinal wall• water leaks into intestinal tract causing diarrhoea

Endotoxin:• Salmonella enters intestinal cells through phagocytosis• endotoxins damage the cells from the inside• cause fever and inflammation in large amounts

Diagnosis

Lactobacillus acidophilus (G +ve)A good bacteria present in everybody’s digestive system

Salmonella (G -ve)A bacteria that causes severe food poisoning

Is the patient severely infected with Salmonella??

Or has he got something else??

You decide!!

GRAM STAIN PROCEDURE

• 1. Gather all equipment and supplies– Gram stain– Microscope slides– Heat source– Sterile swab– Culture or specimen– Microscope

GRAM STAIN PROCEDURE

• 2. Sterilise your bench, wash your hands and light your bunsen burner

GRAM STAIN PROCEDURE

• 3. Choose an isolated colony off of the agar plate and obtain bacteria with a sterile swab.

GRAM STAIN PROCEDURE

• 4. Place the swab on the microscope slide and spread the colonies in a circular motion.

GRAM STAIN PROCEDURE

• 5. Heat fix the microorganisms to the slide by passing the slide through the bunsen flame a couple of times

GRAM STAIN PROCEDURE

• 6. Place slide on staining tray or hold with forceps above the sink.

GRAM STAIN PROCEDURE

• 7. Flood the surface of the slide with Crystal Violet stain and let sit for one minute.

GRAM STAIN PROCEDURE

• 8. Rinse the slide with distilled water.

GRAM STAIN PROCEDURE

• 9. Flood the slide with Gram’s Iodine and time for one minute.

GRAM STAIN PROCEDURE

• 10. Rinse the slide with distilled water.

GRAM STAIN PROCEDURE

• 11. Flood the slide with Gram’s decolorizer and time for 30 seconds.

GRAM STAIN PROCEDURE

• 12. Rinse the slide with distilled water.

GRAM STAIN PROCEDURE

• 13. Flood the slide with the counterstain, Safranin, and let sit for one minute.

GRAM STAIN PROCEDURE

• 14. Rinse the slide with distilled water.

GRAM STAIN PROCEDURE• 15. Blot the slide and read with

the oil immersion lens of the microscope. Look for Gram-negative and Gram-positive bacteria.

Diagnosis

Lactobacillus acidophilus (G +ve)A good bacteria present in everybody’s digestive system

Salmonella (G -ve)A bacteria that causes severe food poisoning

Is Patient No. 986214 severely infected with Salmonella

Or has he just got something else!

You decide!!

Making Yoghurt

Fermentation of milk

which happens when the bacteria in the milk use the milk sugar (lactose).

It is their food source during anaerobic respiration.

The souring of milk is a fermentation process

Lactose lactic acid

Lactobacillus

The milk becomes more and more acidic, lowering the pH and making it taste sour

Product- yoghurt

Yoghurt-making uses 2 species of bacteria to ferment milk (often low-fat). They are added as a starter culture and produce lactic acid.

Lactobacillus acidophilus acidifies the milk

Streptococcus thermophilus turns the milk creamy

When the pH drops to 4.4 the proteins in milk coagulate (thicken) producing yoghurt.

Yoghurt productionMilk products

mixingHeat treatment at 90oC

1.2.3.

Removes oxygen

Kills harmful bacteria

Changes milk proteins

Cooled to 40oC

Best temperature for growing yoghurt bacteria

Yoghurt bacteria added

Ferment milk sugar (lactose) into lactic acid.

Storage tank at 4.5oC

Give yoghurt its acid taste

1.

2.

Fermentation for 6 hours

Slows down further bacterial fermentation

Credit: museumof brands

Make your own!

You will need:• Skimmed UHT milk (this milk is

sterile!)

• Live yoghurt (starter culture)

• Dried milk powder(makes it creamy!

Method

1. Take a clean pot and label with your name and the date

2. Fill 2/3 of the pot with UHT3. Dissolve 1 teaspoon of milk powder into

the milk4. Add 1 teaspoon of yoghurt to the milk5. Test the pH and make a note of it6. Put the lid on and place in the water bath

at 35 degrees C