ISOLATION, GRAM STAINING AND IDENTIFICATION OF BACTERIA BY BY DR. TED PASS II DR. TED PASS II KENTUCKY MICROBIOLOGY LABORATORY KENTUCKY MICROBIOLOGY LABORATORY.

ISOLATION, GRAM STAINING ISOLATION, GRAM STAINING AND IDENTIFICATION OF AND IDENTIFICATION OF

BACTERIABACTERIA BYBY

DR. TED PASS IIDR. TED PASS II

KENTUCKY MICROBIOLOGY KENTUCKY MICROBIOLOGY LABORATORYLABORATORY

CERTIFICATION PROGRAMCERTIFICATION PROGRAM

PROCEDURE FOLLOWED TO PROCEDURE FOLLOWED TO IDENTIFY BACTERIAIDENTIFY BACTERIA

OBTAIN A OBTAIN A PURE CULTUREPURE CULTURE USING A USING A DIFFERENTIAL MEDIA SUCH AS DIFFERENTIAL MEDIA SUCH AS MACCONKEY AGARMACCONKEY AGAR

PREPARE A SLIDE USING PREPARE A SLIDE USING GRAM STAINGRAM STAIN PROCEDUREPROCEDURE

IDENTIFYIDENTIFY BACTERILA ISOLATES BACTERILA ISOLATES USING THE USING THE APIAPI OR OR ENTEROTUBEENTEROTUBE

IDENTIFICATION SYSTEMIDENTIFICATION SYSTEM

MACCONKEY AGAR MacConkey agar is probably the most popular solid differential/selective medium in the world. It is mainly used in isolation of lactose fermenting, Gram-negative enteric pathogens and for inhibiting growth of Gram-positive organisms. Bacterial colonies that can ferment lactose turn the medium red. This red color is due to the pH indicators (Neutral Red) response to the acidic environment created by the fermentation of lactose. MAC is decolorized by NLF bacteria...as they utilize Amino Acids alkaline metabolites are released and the Neutral Red becomes colorless.

Lactose Fermentors vs Non Lactose Fermentors vs Non Lactose FermentorsLactose Fermentors

Examples of Examples of LFLF:: Escherichia coliEscherichia coli Klebsiella pneumoniaeKlebsiella pneumoniae Enterobacter aerogenesEnterobacter aerogenes Citrobacter freundiCitrobacter freundi Examples ofExamples of NLF NLF:: Pseudomonas aeruginosaPseudomonas aeruginosa Proteus mirabilisProteus mirabilis **Gram PositiveGram Positive bacteria are inhibited by Crystal bacteria are inhibited by Crystal

violet and bile salts…i.e., Staphylococcus aureusviolet and bile salts…i.e., Staphylococcus aureus

ISOLATION STREAK TECHNIQUEISOLATION STREAK TECHNIQUE

ISOLATION OF BACTERIA ON MACCONKEYFOR GRAM STAINING AND IDENTIFICATION

Gram-Staining Procedure

The reagents you will need to successfully perform this technique are: •Crystal Violet ( Primary Stain) •Iodine Solution (Mordant) •Decolorizer ( 95%Ethanol ) •Safranin ( Counterstain) •Water (preferably in a squirt bottle)

SMEAR PREPARATION SMEAR PREPARATION

STEP 1 STEP 2

STEP 3 STEP 4

STAINING PROCEDURESTAINING PROCEDURE STEP 1: Flood (cover completely) the STEP 1: Flood (cover completely) the

entire slide with entire slide with Crystal VioletCrystal Violet…CV …CV attaches to the attaches to the cell wallcell wall of both of both G+G+ and and G-G- bacteria. Let the crystal violet bacteria. Let the crystal violet stand for about 60 seconds. stand for about 60 seconds.

STEP 2: Now, flood your slide STEP 2: Now, flood your slide with with

the the iodine solutioniodine solution. . Rinse the slide with water after 60 Rinse the slide with water after 60

sec. At this point, the specimen sec. At this point, the specimen should appear brownish to blue-violet should appear brownish to blue-violet in color. in color. IodineIodine strengthens the bond strengthens the bond between the between the CWCW and the and the CVCV

STEP 3: Add the STEP 3: Add the DecolorizerDecolorizer, , Ethanol for 15 secEthanol for 15 sec. .

To be safe, add the To be safe, add the EthanolEthanol dropwise dropwise until the blue-violet color is no longer until the blue-violet color is no longer emitted from your specimen…emitted from your specimen…Removes Removes 20% lipids20% lipids, i.e., , i.e., LPS and LPS and CV-I complexCV-I complex (Decolorizing the cell) (Decolorizing the cell) Quickly, rinse the slide with the Quickly, rinse the slide with the water for 5 seconds. water for 5 seconds.

STEP 4: The final step involves STEP 4: The final step involves applying the applying the CounterstainCounterstain, ,

SafraninSafranin. . Flood the slide with the dye as you Flood the slide with the dye as you

did in steps 1 and 2. Let this stand did in steps 1 and 2. Let this stand for about a 60 sec. Rinse with water for about a 60 sec. Rinse with water for 5 seconds to remove any excess for 5 seconds to remove any excess dye. dye. SafraninSafranin will attach to will attach to G-G- Cell Cell WallWall

GRAM NEGATIVE AND GRAM GRAM NEGATIVE AND GRAM POSITIVE ORGANISMSPOSITIVE ORGANISMS

GRAM POSITIVE COCCIGRAM POSITIVE COCCI

GRAM NEGATIVE RODSGRAM NEGATIVE RODSGRAM POSITIVE COCCIGRAM POSITIVE COCCI

The Gram stain differentiates between The Gram stain differentiates between bacteria that possess bacteria that possess 2%2% and and 20%20%

Lipids in their cell wall or "cell Lipids in their cell wall or "cell envelope“. The G- CW has an outer envelope“. The G- CW has an outer membrane of Lipopolysaccharides membrane of Lipopolysaccharides

LPSLPS))

ENDOSPORE SLIDEENDOSPORE SLIDE

API IDENTIFICATION SYSTEMAPI IDENTIFICATION SYSTEM This This API-20EAPI-20E test strip test strip (from (from

bioMerieux, Inc.) bioMerieux, Inc.) is used to identify is used to identify the the enteric gram negative rodsenteric gram negative rods; 20 ; 20 separate test compartments are on separate test compartments are on the strip, all dehydrated. A bacterial the strip, all dehydrated. A bacterial suspension is used to rehydrate each suspension is used to rehydrate each of the wells. Some of the wells will of the wells. Some of the wells will have color changes due to pH have color changes due to pH differences: others produce end differences: others produce end products that have to be identified products that have to be identified with reagents. A with reagents. A profile numberprofile number is is determined from the sequence of + determined from the sequence of + and - test results, then looked up in a and - test results, then looked up in a codebookcodebook having a correlation having a correlation between numbers and bacterial between numbers and bacterial speciesspecies . .

INOCULATION OF AN API INOCULATION OF AN API STRIPSTRIP

Prepare a suspension of the Prepare a suspension of the bacteria in the saline tubebacteria in the saline tube

Inoculate a large colony (2-3mm Inoculate a large colony (2-3mm diameter)of the bacterium (diameter)of the bacterium (pure pure cultureculture) into the 0.85% NaCl solution.) into the 0.85% NaCl solution.

Use a Use a McFarlandMcFarland barium sulfate barium sulfate standard #3standard #3

INOCULATIONINOCULATION Holding the strip at a slight angle up from the Holding the strip at a slight angle up from the

table top, you will now inoculate the table top, you will now inoculate the bacterial bacterial suspensionsuspension into each well with the into each well with the sterile pipettesterile pipette. .

Touch the end of the pipette to the side of the Touch the end of the pipette to the side of the cupule,cupule, allowing capillary action to draw the fluid allowing capillary action to draw the fluid into the well as you slowly squeeze the bulb. This into the well as you slowly squeeze the bulb. This should eliminate any bubbles forming in the wells. should eliminate any bubbles forming in the wells. Each well should be filled up to the neck (see Each well should be filled up to the neck (see diagram). diagram).

CIT, VP, and GELCIT, VP, and GEL have boxes around their names. have boxes around their names. These test wells will be filled all the way up to the These test wells will be filled all the way up to the toptop of the well. of the well.

LDC, ODC, ADH, H2S, and URELDC, ODC, ADH, H2S, and URE are filled as are filled as described in step B, but they will then be filled up described in step B, but they will then be filled up to the top with to the top with sterile mineral oilsterile mineral oil. .

TILT AT ANGLE TO FILL TILT AT ANGLE TO FILL CUPULESCUPULES

Incubate the strip in its Incubate the strip in its chamberchamber

The bottom of the The bottom of the incubation chamberincubation chamber has has small indented small indented wellswells in the bottom: fill it with in the bottom: fill it with water just enough to fill these indentations. water just enough to fill these indentations.

Place the strip into this Place the strip into this bottombottom. There should . There should not be so much water that it spills onto the not be so much water that it spills onto the API strip. API strip.

Place the Place the toptop of the incubation chamber over of the incubation chamber over the bottom, and label it. the bottom, and label it.

Place the strip at Place the strip at 3535 to 37 to 37ºº C for C for 18-24 18-24 hours. hours.

• INTERPRETATION:INTERPRETATION: Add the proper reagents to the Add the proper reagents to the

compartments:compartments: 1 drop of 1 drop of Kovac'sKovac's to the to the INDIND (read within a (read within a couple of minutes) couple of minutes)

1 drop of 1 drop of Barritt's A and B to VPBarritt's A and B to VP (a positive (a positive reaction may take up to 10 minutes) reaction may take up to 10 minutes)

1 drop of 1 drop of FeCl3 to TDAFeCl3 to TDA

Record results on the diagram handed Record results on the diagram handed out to you in lab (1, 2, or 4 points for + out to you in lab (1, 2, or 4 points for + reaction, 0 points for - reaction). reaction, 0 points for - reaction). The The oxidase testoxidase test reaction should be reaction should be negative, and is added as the last test negative, and is added as the last test result. result.

Three test reactionsThree test reactions are added together are added together at a time to give a 7-digit number, which at a time to give a 7-digit number, which can then be looked up in the codebook. can then be looked up in the codebook.

ENTEROTUBE IDENTIFICATION ENTEROTUBE IDENTIFICATION SYSTEMSYSTEM

The Enterotube® II contains 12 The Enterotube® II contains 12 different agars enabling the different agars enabling the performance of a total of 15 performance of a total of 15 biochemical tests as well as an biochemical tests as well as an enclosed inoculating wire. enclosed inoculating wire.

of a total of 15 biochemical tests as well as an enclosed inoculating wire.

The Enterotube® II contains 12 different agars enabling the performance

ENTEROTUBE INOCULATIONENTEROTUBE INOCULATION

INOCULATION OF INOCULATION OF ENTEROTUBEENTEROTUBE

1. Remove both caps of the Enterotube® II 1. Remove both caps of the Enterotube® II and with the and with the straight end of the straight end of the inoculatinginoculating wirewire, pick off the equivalent of , pick off the equivalent of a colony from your unknown plate. a colony from your unknown plate. A A visible visible inoculuminoculum should be seen on the should be seen on the tip and side of the wiretip and side of the wire. .

2. 2. InoculateInoculate the Enterotube® II by the Enterotube® II by grasping the grasping the bent-end of the inoculating bent-end of the inoculating wirewire, twisting it, and withdrawing the wire , twisting it, and withdrawing the wire through all through all 1212 compartments using a compartments using a turning motion.turning motion.

ENTEROTUBE (contd.)ENTEROTUBE (contd.)

3. 3. Reinsert the wireReinsert the wire into the tube into the tube (use a turning motion) (use a turning motion) through all 12 through all 12 compartmentscompartments until the until the notchnotch on the on the wire is aligned with the opening of the wire is aligned with the opening of the tube. (The tube. (The tiptip of the wire should be of the wire should be seen in the seen in the citrate citrate compartment.) compartment.) BreakBreak the wire at the notch the wire at the notch by by bending. Do not discard the wire yet. bending. Do not discard the wire yet.

ENTEROTUBE (contd.)ENTEROTUBE (contd.)

4. Using the broken off part of the wire, 4. Using the broken off part of the wire, punch holes through the cellophane punch holes through the cellophane which covers the air inlets located on which covers the air inlets located on the rounded sidethe rounded side of the last 8 of the last 8 compartmentscompartments. Your instructor will show . Your instructor will show you their correct location. Discard the you their correct location. Discard the broken off wire in the broken off wire in the SHARPSSHARPS container. container.

5. 5. Replace both capsReplace both caps and and incubateincubate the the Enterotube® II Enterotube® II on its flat surfaceon its flat surface at 35- at 35-3737°°CC. for 18-24 hours.. for 18-24 hours.

POSITIVE REACTIONSPOSITIVE REACTIONS