# Appendix 10. Calculation and recording sheets

Two calculation and recording sheets have been prepared and included in Appendix 10. They can then be photocopied or used as templates for handwritten sheets or day book entries. Examples of completed recording sheets are also included

Reed Muench calculation sheet

This sheet has been designed to assist with the calculation of the 50 percent endpoint using the Reed Muench method. An example of a completed sheet has also been included. See Section 12 for details about using the Reed Muench method for calculating the infectivity titre.

Microwell plate recording sheet

A recording sheet has also been prepared to assist with recording results of the HA and HI test carried out in microwell plates. Two examples of completed sheets are included.

Example 1: This completed recording sheet shows the results of the first part of the HI test, the titration of the Newcastle disease virus antigen, followed by testing of antigen diluted to give 4 HA units per 25 mL volume.

Example 2: This completed recording sheet shows the results of 8 serum samples tested for Newcastle disease virus antibodies using the HI test.

See Section 11 Serology for more details of the HI assay.

Note: A summary of the results on the loose recording sheets should be entered in the daybook.

For example, record in the day book:

• Results of HA test
• Calculated infectivity titre in EID50/mL.
• Dilution used in the HI test
• HI titres of all the samples tested.

The sheets should then be stored in a file and available for future reference.

Calculation of EID50/mL of virus suspension using Reed and Muench mathematical technique

Name: ......................

Date: ........................

Experiment: ............

 Dilution of Inoculum Include range of dilutions from 100% infected (all HA +ve) to 0% infected (all HA -ve) Number of eggs infected (HA +ve) Number of eggs not infected (HA -ve) Accumulated numbers Percentage infected Infected (A) Not infected (B) Total (A+B) A/(A+B) × 100 ¯ ¯ ¯ ¯ ¯

Determine the dilution of inoculum producing 50 percent infection of eggs.

If necessary, use the Reed Muench formula to calculate the index:

Apply the index calculated using this formula to the dilution that produced the infection rate immediately above 50 percent =

This dilution of the virus suspension contained one EID50 unit of virus in 0.1 mL.

1 mL of the virus suspension will contain ten times the reciprocal of the calculated dilution.

Therefore Infectivity Titre of virus suspension/mL = ...........................

Example of completed sheet calculation of EID50/mL of virus suspension using Reed and Muench mathematical technique

Name: John Smith

Date: 6/6/01

Experiment: Titration of I-2 wet vaccine

 Dilution of Inoculum Include range of dilutions from 100% infected (all HA +ve) to 0% infected (all HA -ve) Number of eggs infected (HA +ve) Number of eggs not infected (HA -ve) Accumulated numbers Percentage infected Infected (A) Not infected (B) Total (A+B) A/(A+B) × 100 10-6 5 ­ 0 ¯ 11 0 11 11/11 = 100% *10-7 4 ­ 1 ¯ 6 1 7 6/7 = 86% 10-8 1 ­ 4 ¯ 2 5 7 2/7 = 29% 10-9 1 ­ 4 ¯ 1 9 10 1/10 = 10% 10-10 0 ­ 5 ¯ 0 14 14 0/14 = 0%

Determine the dilution of inoculum producing 50 percent infection of eggs.

If necessary, use the Reed Muench formula to calculate the index:

Apply the index calculated using this formula to the dilution that produced the infection rate immediately above 50 percent = 10-7.6

(Mark dilution with *)

This dilution of the virus suspension contained one EID50 unit of virus in 0.1 mL.

1 mL of the virus suspension will contain ten times the reciprocal of the calculated dilution.

Therefore Infectivity Titre of virus suspension in EID50/mL = 10 × 107.6 = 108.6 EID50/mL

Example 1: Completed sheet used in HA test

MICROWELL PLATE RECORDING SHEET FOR HI/HA TESTS

 Plate No...................1.................Name..........Jane Smith..............Date:....30.5.01............................ Virus...............Newcastle diseaseRBC...........................1%............................SamplesTitration of I-2 wet vaccine stored at 4°C NotesRBC added at 1200D = dead egg, not tested X = Haemagglutination· = No haemagglutination Sample A 5.4.01 1 2 3 4 5 6 7 8 9 10 11 12 Sample B 3.5.01 A 10-10 A · · · · · · · · · · A B 10-10 A 10-9 B · · X · · · · · · · B B 10-9 A 10-8 C X · · · · · X X · · C B 10-8 A 10-7 D · X X X X X X X X D D B 10-7 A 10-6 E X X X X X X X X X X E B 10-6 A 10-5 F X X X X X X X X X X F B 10-5 RBC control G · · G H H 1 2 3 4 5 6 7 8 9 10 11 12

Note:

One plate has been used for testing allantoic fluid from two sets of eggs.

Set A inoculated with titrated sample from 5.4.01 and Set B inoculated with titrated sample from 3.5.01.

Example 2: Completed sheet used in HI test

MICROWELL PLATE RECORDING SHEET FOR HI/HA TESTS

 Plate No...................1.................Name.......John Smith.................Date:....30.5.01............................ Virus...............Newcastle diseaseRBC...........................1%............................SamplesTitration of antigen for HI testTesting of 4HA units of antigenHI test of standard positive and negative serum using 4HA units of Ag NotesAg = antigenRows A and B RBC added at 1000Ag diluted 1 in 64 with PBSRows E to H, Ag added 1130Rows C to H, RBC added 1200X = Haemagglutination· = No Haemagglutination 1 2 3 4 5 6 7 8 9 10 11 12 ND Ag A X X X X X X X X · · · · A HA titre = 28 = 256 HA units ND Ag B X X X X X X X X · · · · B HA titre = 28 = 256 HA units 1/64 dilution of Ag C X X · · · · · · · · · · C HA titre = 22 = 4 HA units 1/64 dilution of Ag D X X · · · · · · · · · · D HA titre = 22 = 4 HA units Positive serum E · · · · · · X X X X X · E HI titre = 26 Positive serum F · · · · · · X X X X X · F HI titre = 26 Negative serum G X X X X X X X X X X X · G HI titre = 20 Negative serum H X X X X X X X X X X X · H HI titre = 20 1 2 3 4 5 6 7 8 9 10 11 12

Example 3: Completed sheet used in HI test

MICROWELL PLATE RECORDING SHEET FOR HI/HA TESTS

 Plate No.........1..........................Name....Jane Smith...................Date:......11.5.01........................ Virus.....................Newcastle disease...............RBC.................1%.............................................Samples.............. Dose ResponseTrial Serum samples collected 30.1.01............. Notes.Antigen added 1300RBC added 1330...................................................X = Haemagglutination (HA)· = Haemagglutination inhibition (HI) Sample numbers 1 2 3 4 5 6 7 8 9 10 11 12 HI titres log base 2 20 A · · · · X X X X X X X · A 4 25 B · · X X X X X X X X X · B 2 1 C · X X X X X X X X X X · C 1 27 D · · X X X X X X X X X · D 2 36 E · · · X X X X X X X X · E 3 42 F · · · X X X X X X X X · F 3 6 G X X X X X X X X X X X · G 0 55 H · · · · · X X X X X X · H 5 1 2 3 4 5 6 7 8 9 10 11 12

MICROWELL PLATE RECORDING SHEET FOR HI/HA TESTS

 Plate No........................................Name............................................Date:............................................. Virus.......................................................................RBC........................................................................Samples.............................................................................................................................................................................................................................. Notes..................................................................................................................................................................................................................................................... 1 2 3 4 5 6 7 8 9 10 11 12 A A B B C C D D E E F F G G H H 1 2 3 4 5 6 7 8 9 10 11 12